Maleimide-Functionalized PEI 600 Grafted Polyurethane : Synthesis , Nano-Complex Formation with DNA and Thiol-Conjugation of the Complexes for Dual DNA Transfection

A polyurethane (PU) grafted with small molecular weight polyethylenimine (PEI600) was synthesized. This PU-PEI600 can assemble DNA via electrostatic interactions into nano-sized polymer/DNA complexes. The complexes exhibited great transfection efficiency in delivering DNA along with a reduced cell toxicity comparing to commercial PEI25k (Mw ~25,000). In order to establish a system for concurrently delivering two different DNA or RNA molecules for cell reprogramming (e.g., induced pluripotent stem cells) or the necessity of multi-expression (e.g., double knock down), the PU-PEI600 was further functionalized with maleimide molecules. The novel PU-PEI600-maleimide would still effectively interact with assigned DNA and different functions of PU-PEI600-maleimide/DNA complexes were self-conjugated in presence of a dithiol molecule (1,6-hexanedithiol). In this study, two reporter genes (pEGFP-C2 and pLanRFP-N) were used and evidence of green/red fluorescence co-expression in cells was observed. This article brings a new concept and a practical method for a plurality of different DNA molecules that are more efficient to be concurrently delivered and co-expressed. This method is very helpful in studying cellular multi-regulation or in the treatment of disease with multiple gene defects in vivo.


Introduction
Non-viral gene delivery, mostly referred to as polymer-based delivery systems, has been considered potentially safer (low toxicity and immunogenicity) as well as lower restriction of cell specificity in comparison to viral-mediated delivery [1].The optimum of designing synthetic gene delivery vehicles is to build DNA/vehicle assemblies that are biocompatible and efficient enough to be used in human therapy.In our previous reports, cationic polyurethane exhibited high transfection efficiency with relatively low cytotoxicity in vitro and in vivo [2,3].A high molecular weight polyethylenimine (PEI 25k , M w ~25,000) revealed its highly effective potential among vectors based on cationic polymers because of its ability of prominent endolysosomal escape and its character of structural protonation [4].However, these advantages are compromised with its high toxicity that limits its applications to gene therapy.In this study, a cationic polyurethane (PU-PEI 600 ) bearing small molecular weight PEI (M w = 600) in PU's side chains was designed and showed superior capability of transfection efficiency and less cytotoxicity than PEI 25k .So far, nearly all research is dealing with administration of one kind DNA or RNA at a time ("mono transfection").However, in work such as cell reprogramming (e.g., induced pluripotent stem cells) or the need of multi-expression

Polymer Characterization
The structures of synthesized polymers were characterized by nuclear magnetic resonance (NMR, Bruker AMX-400 spectrometer, Berlin, Germany) and Fourier transformed infrared spectroscopy (FT-IR, Mattson Galaxy Series 5000, Middleton, WI, USA).All of the chemical shifts in 1 H NMR spectra were reported in parts per million (ppm).A 99.8% pure dimethylsulfoxide (DMSO-d6) and deuteroxide (D 2 O) were used as the solvent to characterize the polymers.IR spectra were obtained using KBr pellets.The molecular weights of the polymers were determined by gel permeation chromatography analysis (GPC, Waters model LC-2410, Milford, MA, USA) in tetrahydrofuran (THF) after calibration with polystyrene standards.The sample concentration for GPC in THF was 8 mg/mL and the flow rate was 1 mL/min.

Preparation of Plasmid DNA and Cell Culture
Reporter DNA pCMVLacZ, pEEGFP-C2, and pLanRFP-N encode the genes of β-galactosidase, green fluorescent protein, and red fluorescent protein, respectively, were amplified in Escherichia coli (DH5α strain) and purified by column chromatography (Qiagen Plasmid Mega kit, Hilden, Germany) [10].The purified plasmid DNA was dissolved in Tris-EDTA buffer (10 mM Tris and 1 mM EDTA, pH 8.0) and the purity of each DNA was determined by the ratio of UV absorbance at 260/280 nm.The cell line COS-7 (SV 40 virus transformed African green monkey cell line, ATCC CRL-1651) was cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL Co., Ltd., Taiwan) supplemented with 10% fetal bovine serum, penicillin (final 100 IU/mL), streptomycin (final 100 μg/mL) and amphotericin B (final 0.25 μg/mL) at 37 °C in a humidified atmosphere of 5% CO2.

Formation of PU-PEI600/DNA Complexes and PU-PEI600-Maleimide/DNA Complexes
Ten milligrams per milliliter of the polymers were dissolved in 20 mM HEPES buffer (pH 7.4) and serial dilutions were prepared and added to react with DNA (final concentration = 5 μg/mL) for various polymers/DNA ratios (w/w).The polymer/DNA complexes were self-assembled in a HEPES buffer at room temperature for at least 30 min.

Electrophoretic Mobility Assay of Polymer/DNA Complexes
The electrophoretic mobility of the polymer/DNA complexes was measured in a 0.7% agarose gel prepared in Tris-acetate-EDTA (TAE) buffer containing ethidium bromide (0.6 μg/mL).After electrophoresis at 100 V for 30 min, DNA bands were visualized by UV irradiation and photographed.

Preparation of Plasmid DNA and Cell Culture
Reporter DNA pCMVLacZ, pEEGFP-C2, and pLanRFP-N encode the genes of β-galactosidase, green fluorescent protein, and red fluorescent protein, respectively, were amplified in Escherichia coli (DH5α strain) and purified by column chromatography (Qiagen Plasmid Mega kit, Hilden, Germany) [10].The purified plasmid DNA was dissolved in Tris-EDTA buffer (10 mM Tris and 1 mM EDTA, pH 8.0) and the purity of each DNA was determined by the ratio of UV absorbance at 260/280 nm.The cell line COS-7 (SV 40 virus transformed African green monkey cell line, ATCC CRL-1651) was cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL Co., Ltd., Taiwan) supplemented with 10% fetal bovine serum, penicillin (final 100 IU/mL), streptomycin (final 100 µg/mL) and amphotericin B (final 0.25 µg/mL) at 37 ˝C in a humidified atmosphere of 5% CO 2 .
2.6.Formation of PU-PEI 600 /DNA Complexes and PU-PEI 600 -Maleimide/DNA Complexes Ten milligrams per milliliter of the polymers were dissolved in 20 mM HEPES buffer (pH 7.4) and serial dilutions were prepared and added to react with DNA (final concentration = 5 µg/mL) for various polymers/DNA ratios (w/w).The polymer/DNA complexes were self-assembled in a HEPES buffer at room temperature for at least 30 min.

Electrophoretic Mobility Assay of Polymer/DNA Complexes
The electrophoretic mobility of the polymer/DNA complexes was measured in a 0.7% agarose gel prepared in Tris-acetate-EDTA (TAE) buffer containing ethidium bromide (0.6 µg/mL).After electrophoresis at 100 V for 30 min, DNA bands were visualized by UV irradiation and photographed.

Sizes of Polymer/DNA Complexes by Dynamic Light Scattering
The hydrodynamic sizes of polymer/DNA complexes and conjugates of the PU-PEI 600 -maleimide/DNA complexes were determined by dynamic light scattering (DLS) (Nicomp 380 system, San Diego, CA, USA) at 25 ˝C with a 5 mW He-Ne laser (λ = 633 nm) as the incident beam.

XTT Cell Viability Assay for Cell Toxicity
The influence of polymer/DNA complexes and PU-PEI 600 -maleimide/DNA conjugates on cell viability was evaluated in cell culture using the XTT assay [10].COS-7 cells were cultured in complete DMEM with a seeding density of 1 ˆ10 4 cells/well in a 96-well plate.The cells were incubated in an incubator with 5% CO 2 at 37 ˝C for 24 h.Subsequently, the cells were treated with 200 µL FBS-free DMEM containing polymer/DNA complexes or PU-PEI 600 -maleimide/DNA conjugates.After incubation for 1 h with the polymer/DNA complexes or the conjugates, the cells were washed with 200 µL PBS and replaced by complete DMEM for further 48 h incubation.Then, 50 µL of XTT reagent was added to each well and incubated at 37 ˝C for 1 h.The results were expressed as the relative cell viability (%) with respect to control wells containing cells that were only treated with DMEM.

Transfection Protocol for Cells Delivered with pCMV-LacZ DNA by ONPG Assay
COS-7 cells were used to evaluate the transfection efficiency of PU-PEI 600 /DNA complexes and PU-PEI 600 -maleimide/DNA complexes.The cells in complete DMEM were seeded in a 96-well plate (1 ˆ10 4 cells per well) and incubated for 24 h before transfection.The DNA concentration was kept constant at 5 µg/mL (1 µg/well) and the amounts of polymers were varied for designated polymer/DNA ratios.Two-hundred-microliter solutions of the complexes were taken and incubated with cells for 1 h at 37 ˝C.For the diluted experiments of dual transfection, 4-fold dilution of these complexes was made in DMEM before cell incubation.The medium was replaced afterwards with complete DMEM and the cells were incubated for further 48 h.For evaluating transfection efficiency, the cells were washed with 0.3 mL PBS and then permeabilized with 20 µL cell lysis buffer at 4 ˝C for 20 min.An ONPG solution (180 µL/well) was added after lysis treatment and the cells were incubated at 37 ˝C for another 1 h.The expression of pCMV-LacZ gene was measured spectrometrically using an Enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 405 nm.

Dual DNA Transfection via Thiol-Conjugates of PU-PEI 600 -Maleimide/DNA Complexes
For dual DNA transfection, two distinct PU-PEI 600 -maleimide/pEEGFP-C2 complexes and PU-PEI 600 -maleimide/pLanRFP-N complexes were prepared as the method stated in experimental section.To obtain conjugates of PU-PEI 600 -maleimide/DNA complexes, these two different PU-PEI 600 -maleimide/DNA complexes were reacted with 1.6-hexanedithiol dissolved in HEPES buffer via a reaction between maleimide and SH [11,12].The thiol-conjugated reaction was conducted for 30 min at room temperature and the condition (as an optimal molar ratio of PU-PEI 600 -maleimide/dithiol) for preparation of the conjugates was studied.
The cells were seeded in a 24-well plate (6 ˆ10 4 cells per well) in complete DMEM as a culture medium and incubated for 24 h before transfection.An amount of 1200 µL cell medium containing the conjugates of two distinct PU-PEI600-maleimide/DNA complexes was taken and incubated with cells for 1 h at 37 ˝C.The medium was replaced afterwards with complete DMEM and the cells were incubated for further 48 h.For evaluation of dual DNA transfection efficiency, the co-expression of pEEGFP-C2 (green fluorescence protein, GFP) and pLanRFP-N (red fluorescence protein, RFP) was observed using fluorescence (Nikon ECLIPSE TE2000-U, Tokyo, Japan) microscope under two magnifications of 100ˆand 400ˆ.

Results and Discussion
3.1.Synthesis and Characterization of PU-PEI 600 and PU-PEI 600 -Maleimide PU-PEI 600 and PU-PEI 600 -maleimide were synthesized as shown in Schemes 1 and 2. The chemical structures of these polymers were confirmed by FT-IR and 1 H NMR spectroscopy.The peaks in the FT-IR spectra at 1720 cm ´1 (C=O stretching, urethane), 1651 cm ´1 (C=O stretching, amide), 1553 cm ´1 (N-H bending, amide), and 3320 cm ´1 (N-H stretching, urethane) represent the absorption of urethane links in PU-PEI 600 and PU-PEI 600 -maleimide.There was no obvious difference in FT-IR spectra between PU-PEI 600 and PU-PEI 600 -maleimide because only a few maleimide groups were grafted in PU-PEI 600 (see below).The chemical shifts of characterized protons of PU-PEI 600 and PU-PEI 600 -maleimide are reported in the experimental section on polymer synthesis.PU-PEI 600 was formed as evidenced by disappearance of the characteristic peak of CH(COO)-(3.4 ppm) from PU and appearance of the peak of PEI, -CH-CH-(2.48-3.14ppm), in PU-PEI 600 .Furthermore, the synthesized PU-PEI 600 -maleimide was identified by a particular peak of maleimide of -C=C-(8.22 ppm).The GPC data showed that the weight averaged molecular weights of PU and PU-PEI 600 were 15,428 and 36,413, respectively with a polydispersity range from 1 to 1.15.The increase in molecular weight indicates that about 30 PEI 600 molecules have been successfully connected to the side chains of PU.On the other hand, small difference between PU-PEI 600 and PU-PEI 600 -maleimide (M w ~37,306) suggested that only a few maleimide groups were grafted on PU-PEI 600 (calculated as 4-6 maleimide molecules grafted on a PU-PEI 600 structure).

The Gel Retardation and Size Analysis of PU-PEI 600 /DNA Complexes
The ability of cationic polymers interacting electrostatically with DNA is positively related to a reduction in DNA electrophoretic mobility.Therefore, the relative ability of polymers to do DNA condensation into complexes can be assessed by comparing the polymer/DNA ratios where free DNA band is still observed.It can be seen in Figure 1A that at 1/1 ratio (w/w) of PU-PEI 600 /DNA, the disappearance of DNA bands starting from lane 2 suggests that amine groups of PU-PEI 600 with positive charges can effectively interact with DNA to form complexes.It is worth noting that commercial PEI 25k also acts to retain DNA in their complexes at 1/1 weight ratio [13,14].Since a very similar structure is shared in both outer shell of PEI 25k and the side chains of PU-PEI 600 , respectively, the difference in the "interior" polymer's structures having little contribution to DNA condensation is also suggested.However, the unlikeness in polymer's structure (e.g., backbone) may change the profile of buffering capacity due to different amount of total amines that react in acid titration (see Supplementary Materials).
The size of polymer/DNA complexes is influenced by a polymer capable of doing DNA condensation.Figure 1B shows the size of PU-PEI 600 /DNA complexes at ratios ranging from 1/1 to 100/1 (w/w).The average diameters of these complexes were within 100-200 nm at ratios starting from 5/1 (w/w) and were small enough for cellular endocytosis (<0.2 µm) [15].The discrepancy in the results at weight ratios between DNA condensation (at 1/1) and size measurement (at 5/1) says that noticeable DNA condensation starts early before smallest polymer/DNA complexes were formed.Since the size of polymer/DNA complexes would be affected by polymer's structural arrangement (including backbone structure of polymers) [16,17] and density distribution of positive charges [18], the size difference in the complexes using PEI 600 in comparison with PU-PEI 600 was observed (Figure 1B).Therefore, the size of polymer/DNA complexes is more informative for describing the extent of polymer/DNA interactions than DNA electrophoretic mobility.

The Cytotoxicity and Transfection Assay of PU-PEI600/DNA Complexes
The cytotoxicity is an important factor for a success of gene delivery.To determine the cytotoxicity of PU-PEI600/DNA in comparison with that of PEI600/DNA, cells were incubated with increasing amounts of PU-PEI600/DNA or PEI600/DNA complexes at fixed DNA concentration (5 μg/mL) and relative viability of these complexes was measured.As shown in Figure 2A, PU-PEI600/DNA complexes exhibited no toxicity on COS-7 cells till a ratio of 100/1 (w/w) was reached.The PEI600/DNA complexes also exhibited no toxicity as PU-PEI600/DNA complexes.This phenomenon is attributed to the use of low molecular weight PEI [19] since a higher molecular weight polymer with dense positive charges (e.g., PEI25k) would result in a prominent cytotoxicity due to a great extent of disturbance on cell membrane.
The transfection efficiency of complexes in delivering pCMV-LacZ DNA was evaluated by the expression of β-galactosidase (equivalent to ONPG absorbance).The effect of ratios of PU-PEI600/DNA and PEI600/DNA complexes on their transfection efficiency in COS-7 cells are shown in Figure 2B.PU-PEI600 with a polymer structure composed of PEI600 (Mw ~36000) bears a great transfection potential.In contrast, the use of PEI600 (Mw = 600) barely resulted in any DNA expression even though a capability of condensation with DNA into small complexes by PEI600 was noted (Figure 1B).This is because of early dissociation between small cationic molecules (PEI600) and DNA in cellular compartments and limited stability of the formed complexes [20].The best transfection efficiency of PU-PEI600/DNA complexes was reached at a broad range of ratios from 5/1 to 20/1 (w/w), which is in discrepancy to PEI25k/DNA complexes in which a sharp change occurred before/after its optimal ratio

The Cytotoxicity and Transfection Assay of PU-PEI 600 /DNA Complexes
The cytotoxicity is an important factor for a success of gene delivery.To determine the cytotoxicity of PU-PEI 600 /DNA in comparison with that of PEI 600 /DNA, cells were incubated with increasing amounts of PU-PEI 600 /DNA or PEI 600 /DNA complexes at fixed DNA concentration (5 µg/mL) and relative viability of these complexes was measured.As shown in Figure 2A, PU-PEI 600 /DNA complexes exhibited no toxicity on COS-7 cells till a ratio of 100/1 (w/w) was reached.The PEI 600 /DNA complexes also exhibited no toxicity as PU-PEI 600 /DNA complexes.This phenomenon is attributed to the use of low molecular weight PEI [19] since a higher molecular weight polymer with dense positive charges (e.g., PEI 25k ) would result in a prominent cytotoxicity due to a great extent of disturbance on cell membrane.
The transfection efficiency of complexes in delivering pCMV-LacZ DNA was evaluated by the expression of β-galactosidase (equivalent to ONPG absorbance).The effect of ratios of PU-PEI 600 /DNA and PEI 600 /DNA complexes on their transfection efficiency in COS-7 cells are shown in Figure 2B.PU-PEI 600 with a polymer structure composed of PEI 600 (M w ~36000) bears a great transfection potential.In contrast, the use of PEI 600 (M w = 600) barely resulted in any DNA expression even though a capability of condensation with DNA into small complexes by PEI 600 was noted (Figure 1B).This is because of early dissociation between small cationic molecules (PEI 600 ) and DNA in cellular compartments and limited stability of the formed complexes [20].The best transfection efficiency of PU-PEI 600 /DNA complexes was reached at a broad range of ratios from 5/1 to 20/1 (w/w), which is in discrepancy to PEI 25k /DNA complexes in which a sharp change occurred before/after its optimal ratio at 1/1 (data not shown).This observation demonstrates that grafting of short PEI 600 into the side chain of PU (as PU-PEI 600 ) has created a great improvement in the transfection profile in comparison with PEI 25k or PEI 600 (see Figure 2C).
Polymers 2015, 7, page-page 8 at 1/1 (data not shown).This observation demonstrates that grafting of short PEI600 into the side chain of PU (as PU-PEI600) has created a great improvement in the transfection profile in comparison with PEI25k or PEI600 (see Figure 2C).PU-PEI 600 was further modified to carry maleimide groups via a between the amine group of PU-PEI 600 and maleimide-NHS.A similarity in the profiles of DNA condensation and sizes between PU-PEI 600 -maleimide (Figure 3A,B) and PU-PEI 600 (Figure 1A,B) is shown.These results are in agreement with FT-IR, NMR and GPC data that only a few maleimide structures were grafted on PU-PEI 600 and therefore, had minor influence on DNA condensation and the complex sizes.However, as shown in Figure 3C the optimal ratio of PU-PEI 600 -maleimide/DNA complexes for the best transfection was slightly shifted to 10/1 (from PU-PEI 600 /DNA at 5/1; w/w).The rational explanation is that less amine groups available for protonation after maleimide modification and the introduction of hydrophobic property of maleimide groups into the polymer structure.

The Gel Retardation, Size Analysis and Transfection Efficiency of PU-PEI600-Maleimide/DNA Complexes
PU-PEI600 was further modified to carry maleimide groups via a reaction between the amine group of PU-PEI600 and maleimide-NHS.A similarity in the profiles of DNA condensation and sizes between PU-PEI600-maleimide (Figure 3A,B) and PU-PEI600 (Figure 1A,B) is shown.These results are in agreement with FT-IR, NMR and GPC data that only a few maleimide structures were grafted on PU-PEI600 and therefore, had minor influence on DNA condensation and the complex sizes.However, as shown in Figure 3C the optimal ratio of PU-PEI600-maleimide/DNA complexes for the best transfection was slightly shifted to 10/1 (from PU-PEI600/DNA at 5/1; w/w).The rational explanation is that less amine groups available for protonation after maleimide modification and the introduction of hydrophobic property of maleimide groups into the polymer structure.In order to achieve dual transfection into a cell, conjugation of two distinct polymer/DNA complexes was designed.PU-PEI 600 -maleimide/DNA1 and PU-PEI 600 -maleimide/DNA2 complexes were individually prepared at PU-PEI 600 -maleimide/DNA ratio of 10/1 (see Section 3.4.and Figure 3C).The maleimide groups of these two different complexes can be conjugated via a presence of 1,6-hexanedithiol as a thioether linker.Figure 4A shows the effect of 1,6-hexanedithiol on the size of the conjugates of PU-PEI 600 -maleimide/DNA complexes.An increase in the size of the conjugates was detected as an increased amount of the dithiol was added.Our result also shows the average size of the conjugates of PU-PEI 600 -maleimide/DNA complexes was about 200 nm when a molar ratio at 1.25/1 of dithiol/PU-PEI 600 -maleimide was applied.Since the average size of PU-PEI 600 -maleimide/DNA complexes (at w/w ratio of 10/1) was below 100 nm (Figure 3B), it can be estimated that a conjugate (at 1.25/1 molar ratio of dithiol/PU-PEI 600 -maleimide-DNA) could consist of 2-3 PU-PEI 600 -maleimide/DNA complexes and the probability to have two different kinds of complexes in a conjugate is estimated as 50% and 75%, respectively.
The cytotoxicity of 1,6-hexanedithiol and the conjugates of PU-PEI 600 -maleimide/DNA complexes containing the dithiol was determined by XTT assay.Figure 4B shows that the cytotoxicity of these conjugates is dependent to the increased dithiol added for thiol-conjugation.It is clear that there was more than 90% cell viability when dithiol/PU-PEI 600 -maleimide-DNA molar ratios were below 5/1.

The Effect of Thiol-Conjugation on the Size and Cytotoxicity of PU-PEI600-Maleimide/DNA Complexes
In order to achieve dual DNA transfection into a cell, conjugation of two distinct polymer/DNA complexes was designed.PU-PEI600-maleimide/DNA1 and PU-PEI600-maleimide/DNA2 complexes were individually prepared at PU-PEI600-maleimide/DNA ratio of 10/1 (see Section 3.4.and Figure 3C).The maleimide groups of these two different complexes can be conjugated via a presence of 1,6-hexanedithiol as a thioether linker.Figure 4A shows the effect of 1,6-hexanedithiol on the size of the conjugates of PU-PEI600-maleimide/DNA complexes.An increase in the size of the conjugates was detected as an increased amount of the dithiol was added.Our result also shows the average size of the conjugates of PU-PEI600-maleimide/DNA complexes was about 200 nm when a molar ratio at 1.25/1 of dithiol/PU-PEI600-maleimide was applied.Since the average size of PU-PEI600-maleimide/DNA complexes (at w/w ratio of 10/1) was below 100 nm (Figure 3B), it can be estimated that a conjugate (at 1.25/1 molar ratio of dithiol/PU-PEI600-maleimide-DNA) could consist of 2-3 PU-PEI600-maleimide/DNA complexes and the probability to have two different kinds of complexes in a conjugate is estimated as 50% and 75%, respectively.
The cytotoxicity of 1,6-hexanedithiol and the conjugates of PU-PEI600-maleimide/DNA complexes containing the dithiol was determined by XTT assay.Figure 4B shows that the cytotoxicity of these conjugates is dependent to the increased dithiol added for thiol-conjugation.It is clear that there was more than 90% cell viability when dithiol/PU-PEI600-maleimide-DNA molar ratios were below 5/1.

Cellular Delivery of pCMV-lacZ DNA via PU-PEI600-Maleimide after Thiol-Conjugation
In order to find out an optimal condition for thiol-conjugation, the PU-PEI600-maleimide/DNA (pCMV-lacZ) complexes were conjugated with various concentration of the dithiol and transfection In order to find out an optimal condition for thiol-conjugation, the PU-PEI 600 -maleimide/DNA complexes were conjugated with various concentration of the dithiol and transfection efficiency of the conjugates was evaluated by ONPG absorbance.As can be seen in Figure 5A, the transfection efficiency of PU-PEI 600 -maleimide/DNA complexes without the dithiol was only marginally higher than the conjugates of PU-PEI 600 -maleimide/DNA with a molar ratio of dithiol/PU-PEI 600 -maleimide-DNA at 1.25/1.The reason for this slight reduction of transfection efficiency is due to a slight increase in particle size of the conjugates (~0.2 µm) (Figure 4A) and less favorable for cellular endocytosis.
Polymers 2015, 7, page-page 11 efficiency of the conjugates was evaluated by ONPG absorbance.As can be seen in Figure 5A, the transfection efficiency of PU-PEI600-maleimide/DNA complexes without the dithiol was only marginally higher than the conjugates of PU-PEI600-maleimide/DNA with a molar ratio of dithiol/PU-PEI600-maleimide-DNA at 1.25/1.The reason for this slight reduction of transfection efficiency is due to a slight increase in particle size of the conjugates (~0.2 μm) (Figure 4A) and less favorable for cellular endocytosis.

Dual Transfection of pEGFP-C2 and pLanRFP-N DNA via the Conjugates of PU-PEI600-Maleimide/DNA Complexes
In this study, two varieties of DNA (pEGFP-C2 and pLanRFP-N) encoding green and red fluorescence protein (GFP and RFP, respectively) were used to study the hypothesis of co-expression of different PU-PEI600-maleimide/DNA complexes via the thiol-conjugation.After PU-PEI600-maleimide/pEGFP-C2 complexes and PU-PEI600-maleimide/pLanRFP-N complexes (both prepared at an optimal weight ratio of 10/1, Figure 3C) were prepared, the conjugates, at PU-PEI600-maleimide-DNA/dithiol molar ratio of 1/1.25 can be formed to have ideally small particles (Figure 5A).The conjugates mainly contain two to three PU-PEI600-maleimide/DNA complexes with 50% to 75% chance of enclosing two distinct DNA, respectively.The efficiency of dual DNA transfection of the conjugates is expressed by showing concurrent GFP and RFP expression in a COS-7 cell under fluorescence observation.
Figure 5B,C shows the fluorescent images of co-expression of GFP (left) and RFP (right) in many cells transfected by the conjugates of PU-PEI600-maleimide/DNA complexes.Remarkably, a substantial percentage of cells showed both expression of GFP and RFP (Figure 5B).Traditionally,  In this study, two varieties of DNA (pEGFP-C2 and pLanRFP-N) encoding green and red fluorescence protein (GFP and RFP, respectively) were used to study the hypothesis of co-expression of different PU-PEI 600 -maleimide/DNA complexes via the thiol-conjugation.After PU-PEI 600 -maleimide/pEGFP-C2 complexes and PU-PEI 600 -maleimide/pLanRFP-N complexes (both prepared at an optimal weight ratio of 10/1, Figure 3C) were prepared, the conjugates, at PU-PEI 600 -maleimide-DNA/dithiol molar ratio of 1/1.25 can be formed to have ideally small particles (Figure 5A).The conjugates mainly contain two to three PU-PEI 600 -maleimide/DNA complexes with 50% to 75% chance of enclosing two distinct DNA, respectively.The efficiency of dual Figure 5B,C shows the fluorescent images of co-expression of GFP (left) and RFP (right) in many cells transfected by the conjugates of PU-PEI 600 -maleimide/DNA complexes.Remarkably, a substantial percentage of cells showed both expression of GFP and RFP (Figure 5B).Traditionally, for the intention of dual DNA transfection, cells can be treated by a mix of these different PU-PEI 600 -maleimide/DNA complexes without the dithiol [21].We knew that about 20% of cells can be transfected and express either GFP or RFP by "mono transfection" of PU-PEI600-maleimide/pEGFP-C2 complexes or PU-PEI 600 -maleimide/pLanRFP-N complexes, respectively.Theoretically, however, the probability of having simultaneous GFP and RFP co-expression in a cell is far less around 4% (=20% ˆ20%), as shown in Figure 5D.Indeed, the level of co-expression would be enhanced using a mix of two concentrated polymer/DNA complexes but, on the other hand, this treatment would result in an increase in cytotoxicity [22].Another clear demonstration is shown (Figure 5E,F) when cells were treated with the four-fold diluted conjugates (Figure 5E) comparing to a mix of two PU-PEI 600 -maleimide/DNA complexes after four-fold dilution (Figure 5F).The latter method showed that, without conjugation, the co-expression of both PU-PEI 600 -maleimide/DNA complexes was very low (Figure 5F).Since several DNA molecules could be incorporated into a particle of formed complexes [23], another comparison was also included in which PU-PEI 600 -maleimide was in complexation with a mix of two different DNA without diSH before adding into cells.Again, the result showed an unbalanced DNA transfection and the co-expression of both GFP and RFP was very low (Figure 5G).Taken together, the use of the conjugates of two different PU-PEI 600 -maleimide/DNA complexes would greatly increase the success rate of dual DNA transfection into a cell with good biocompatibility.

Conclusions
A new polymer, PU-PEI 600 -maleimide, was synthesized.The transfection efficiency of PU-PEI 600 -maleimide/DNA complexes in COS-7 cells was much better at delivering a pCMV-lacZ DNA than short chain PEI 600 .For dual DNA transfection, the PU-PEI 600 -maleimide/DNA complexes containing two different kinds of DNA were thiol-conjugated.This practical method shows that a plurality of different DNA molecules is more efficient to be concurrently delivered and co-expressed.The cell compatibility accompanied by the dual DNA transfection was also remarkable.This method, therefore, is very helpful in studying cellular multi-regulation or in the treatment of disease with multiple gene defects in vivo.

Figure 5 .
Figure 5. (A) Transfection efficiency of the conjugates of PU-PEI 600 -maleimide/DNA complexes at various diSH/PU-PEI 600 -maleimide-DNA molar ratios.Results are presented as mean ˘SD (n = 3); (B,C) The dual DNA transfection of the conjugates of two different PU-PEI 600 -maleimid/DNA complexes after thio conjugation and 100ˆand 400ˆmagnification, respectively; (D) Transfection of a mix of two different PU-PEI 600 -maleimide/DNA complexes without dithiol conjugaion (100ˆ); (E) The dual DNA transfection of the four-fold diluted conjugates of two different PU-PEI 600 -maleimid/DNA complexes; (F) Transfection of a four-fold diluted mix of two different PU-PEI 600 -maleimide/DNA complexes without dithiol conjugaion; (G) Transfection of PU-PEI 600 -maleimide in complexation with a mix of two DNA after four-fold dilution without dithiol conjugaion.

Polymers 2015, 7 ,
2131-2145 DNA transfection of the conjugates is expressed by showing concurrent GFP and RFP expression in a COS-7 cell under fluorescence observation.