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J Mater Sci: Mater Med (2012) 23:2967–2980 DOI 10.1007/s10856-012-4753-9

Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studies Alireza Nouri • Rita Castro • Visvaldas Kairys Jose´ L. Santos • Joa˜o Rodrigues • Yulin Li • Helena Toma´s



Received: 10 June 2012 / Accepted: 22 August 2012 / Published online: 4 September 2012 Ó Springer Science+Business Media, LLC 2012

Abstract In the present study, the effect of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto branched poly(ethylenimine) (PEI) with different grafting degree was examined for gene delivery applications. The DMAEMA-grafted-PEI conjugates were characterized and complexed with plasmid DNA (pDNA) at various concentrations, and the physicochemical properties, cell viability, and in vitro transfection efficiency of the complexes were evaluated in HEK 293T cells. Computational techniques were used to analyze the interaction energies and possible binding modes between DNA and conjugates at different grafting degrees. The cytotoxicity analysis and in vitro transfection efficiency of the conjugate/pDNA complexes exhibited a beneficial effect of DMAEMA conjugation when compared to PEI alone. The computational results revealed that the DNA/vector interaction energy decreases with increasing grafting degree, which can be associated to an enhanced release of the pDNA from the carrier once inside cells. The results indicate the

A. Nouri  R. Castro  J. L. Santos  J. Rodrigues  Y. Li (&)  H. Toma´s (&) CQM—Centro de Quı´mica da Madeira, MMRG, Universidade da Madeira, Campus da Penteada, 9000-390 Funchal, Portugal e-mail: [email protected] H. Toma´s e-mail: [email protected] V. Kairys Department of Bioinformatics, Institute of Biotechnology, Vilnius University, Graicˇiu¯no 8, 02241 Vilnius, Lithuania Present Address: J. L. Santos Department of Materials Science and Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA

significance of DMAEMA conjugation onto PEI as a promising approach for gene delivery applications.

1 Introduction In order to genetically modify a eukaryotic cell, the recombinant DNA has to enter the cell through the cellular membrane, and finally enter the nucleus, where transcription takes place [1]. Although viral vectors are known to be extremely efficient in gene delivery, nonviral methods exhibit certain advantages over viral ones, such as low cost, stability, simple large-scale production, and low host immunogenicity [2]. Thus, there is a fundamental requirement for the development of novel non-viral vectors to condense plasmid DNA (pDNA) that could efficiently deliver the target gene to cells with minimum toxicity [3]. One of the most promising classes of non-viral vectors for gene therapy is formed by cationic polymers, also known as polycations. Through electrostatic interactions between the positively charged amino groups of polycations and the negatively charged phosphate groups of pDNA, these polymers are able to condense pDNA molecules in small sized nanoparticles and form polymer/pDNA complexes (i.e. polyplexes) with a neutral or positive net charge [4]. Polycations are particularly attractive because of their potential safety, nucleic acid cargo capacity, and design ability [5, 6]. Poly(ethylenimine) (PEI) in its high-molecular weight branched form is amongst the most efficient synthetic polycations used in gene delivery research, containing hydrocarbon chains with primary, secondary, and tertiary amino groups. Its positive charge at neutral and acidic pH makes PEI a pDNA condensing and endosomal disruption agent [7]. However, PEI is not the ideal transfection agent

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because of its overwhelming cytotoxicity, thought to be related with a high molecular weight and charge density, conformational flexibility, and type of amino groups (primary amino groups) along the structure [8]. To overcome this problem, several modification strategies have been used including conjugation of biocompatible molecules onto PEI backbone [9, 10]. Polymers of DMAEMA (pDMAEMA) alone and their co-polymers have been used in the delivery of pDNA into eukaryotic cells with promising results [11–13]. Although some researchers have compared the transfection efficiency and cellular uptake mechanism of PEI with pDMAEMA [1, 14, 15], to best of our knowledge, no one has yet reported the effect of DMAEMA conjugation onto PEI (DMAEMAg-PEI) at varying grafting degrees and the subsequent application of the resultant conjugates in gene delivery. Thus, the present study attempts to study the role of DMAEMA conjugation in improving PEI-mediated gene delivery. Herein, the effect of substituting the primary amines of PEI by secondary and tertiary amines is evaluated by reaction of N,N-dimethylaminoethyl methacrylate (DMAEMA) with PEI. The complexes formed by the polymers and pDNA (polyplexes) were characterized in terms of size and zeta potential, and docking studies were performed to better understand the interactions established between the molecules involved. The effects of polymer concentration and grafting degree on transfection efficiency and cytotoxicity were studied on a popular immortalized cell line, namely human embryonic kidney (HEK 293T) cells.

2 Materials and methods 2.1 Materials Branched poly(ethylenimine) (Mw 25 kDa) was purchased from Sigma-Aldrich. The polymer was solubilized in HClacidified water (1 mg/mL), neutralized with NaOH, sterilized by filtration (0.22 lm) and kept at -20 °C. N,Ndimethylaminoethyl methacrylate (99 %, Mw 157.2 g/mol) was purchased from Acros Organics. Reporter lysis buffer (RLB) and luciferase assay system were from Promega. All other reagents and kits used in the work, unless otherwise stated, were obtained from Sigma-Aldrich. 2.2 Amplification of plasmid DNA A plasmid DNA (pDNA) encoding for Enhanced Green Fluorescent Protein and luciferase (pEGFPLuc, 6.4 kb) with a cytomegalovirus promoter (CMV) was used. The plasmids were amplified in Escherichia Coli host strain,

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DH5a, grown overnight in Luria-Broth Base medium containing antibiotic. Afterwards, the plasmids were isolated and purified via GenEluteTM Endotoxin-Free Plasmid maxiprep kit. The concentration of pDNA was assessed at wavelengths of 260 and 280 nm (OD260/OD280 1.8 or greater) using a Cintra 40 UV–Visible Spectrometer (GBC Scientific Equipment). pDNA was stored in pure water at -20 °C until used. 2.3 Synthesis and conjugation of DMAEMA-g–PEI The grafting of DMAEMA monomers onto PEI backbone was accomplished by Michael addition reaction. PEI (0.5 g) was dissolved in 3 mL of methanol in an ice bath. Varying feed amounts of DMAEMA monomer (40, 180, and 360 lL) were added drop-wise to the PEI solution, followed by N2 purge for 10 min. The reaction was carried out at room temperature for 72 h under N2 atmosphere. The crude products were further purified by dialysis against distilled water for 3 days using a Spectra/Por membrane (MWCO = 12,000). After dialysis, the pH of the solution was adjusted to 4–5 using 1 M HCl according to Ref [16]., and the final solution was lyophilized to obtain the product. 2.4 Characterization of DMAEMA-g-PEI 1

H nuclear magnetic resonance (NMR) spectra were taken at RT using a Brucker 400 MHz Avance II? NMR spectrometer. Unmodified and grafted PEIs were dissolved in D2O. The grafting degree was determined from the ratio between the peaks of PEI (d = 2.6–3.6 ppm) and methyl of DMAEMA (d = 1.1 ppm). 2.5 Acid–base titration The buffering capacity of the unmodified and grafted PEIs was determined by acid–base titration assay over the pH values ranging from 2 to 10. First, the unmodified and grafted PEIs were diluted to a final concentration of 1 mg/ mL by adding 10 mg of each polymer in 10 mL of NaCl (150 mM) aqueous solution. Subsequently, the pH of the solution was adjusted to 2.0 by 1 M HCl before titration proceeded. A 100 lL aliquot of 0.1 M NaOH was successively added to the above polymer solutions, and the changes in pH were monitored by a pH meter (model 744, Metrohm). 2.6 Measurements of the size and zeta-potential of the polymer/DNA complexes Dynamic light scattering (DLS) with a Zetasizer Nano-ZS (Malvern) was used to measure the particle size and zeta potential of the unmodified and grafted PEI polyplexes

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formed with pDNA at 25 °C. First, 5 lg of pDNA was added in each microcentrifuge tube and diluted using pure water (Milli-Q, Millipore). Then, appropriate volumes of DMAEMA-g-PEI and PEI aqueous solutions (0.5 mg/mL) were added to the above solution to make measurements at different polymer/pDNA ratios. The total volume for each sample was 800 lL. The resultant mixtures were vortexed for 30 s and incubated at ambient temperature for 10 min prior to the measurement. The particle size of the freshly prepared complexes was measured at 25 °C with a scattering angle of 173°. The zeta potential was determined using a standard capillary electrophoresis cell of Zetasizer Nano ZS with a detection angle of 17° at 25 °C. All the average values were performed with the data from three separate measurements. 2.7 PicoGreen intercalation assay Interaction between pDNA and the polycation was determined using the PicoGreenÒ (Invitrogen) assay. PicoGreenÒ is a sensitive fluorescent stain (fluorochrome) used to quantify double-stranded DNA in solution. Upon binding with free DNA, the fluorescence intensity of PicoGreenÒ increases significantly. The larger the amount of DNA in solution, the higher the fluorescence intensity. Complexes between pDNA and different concentrations (0–10 lg/mL) of unmodified and grafted PEIs were prepared in pure water using 0.2 lg of pDNA in a final volume of 100 lL and further incubated for 20 min at room temperature. Meanwhile, PicoGreenÒ reagent was diluted (200-fold dilution) in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) and 100 lL of the solution were added to each complex. The resultant mixtures were incubated for 5 min at room temperature and protected from the light. The mixtures were added to an opaque 96-well plate and PicoGreenÒ fluorescent emission was measured in a microplate reader (model Victor3 1420, PerkinElmer) at kex = 485 nm, kem = 535 nm. The measurements were carried out in triplicates and the relative fluorescence percentage (%F) was determined using the following equation: %F ¼

Fcomplex  Fblank  100 Ffree DNA  Fblank

The fluorescence from free DNA was considered 100 %. 2.8 Cell lines and cell cultures The experiments described here were performed in vitro on human embryonic kidney 293T (HEK 293T). The cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10 % fetal bovine serum (FBS), and 1 % Antibiotic and Antimycotic solution

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(AbAm) (all purchased from Gibco) at 37 °C in a humidified atmosphere containing 5 % CO2. 2.9 Preparation of the polymer/DNA complexes Complexes of unmodified and grafted PEIs with pDNA were first formed by diluting pDNA (1.5 lg) with pure water in a 1.5 mL microcentrifuge tube. Subsequently, an appropriate amount of the polymer solution (0.5 mg/mL) was added drop-wise to the above solution so as to reach the total volume of 100 lL. The polymer and water were added in such amount to yield the final concentrations of 5, 10 and 20 lg/mL in each well while the concentration of pDNA was kept constant. The tube was capped immediately following the addition of the polymers and vortexed for 30 s. The mixtures were then allowed to incubate at ambient temperature for 10 min, before being added to the cell culture. 2.10 Cytotoxicity studies Cytotoxicity of unmodified and grafted PEIs polyplexes was evaluated 24 h post-transfection on HEK 293T cell line. Cytotoxicity was evaluated by determining the percentage of cell viability (in respect to unexposed cells) using the resazurin reduction assay that establishes a correlation between the cellular metabolic activity and the number of viable cells in culture [17]. The resazurin reagent was prepared by mixing 0.01 g of resazurin with 100 mL of Dulbecco’s phosphate buffered saline (DPBS, 19). For each 100 lL of medium present in the well, 10 lL of resazurin reagent was added and the well plates were incubated for 3 h in a humidified atmosphere with 5 % CO2 at 37 °C. Subsequently, the resultant medium was transferred to 96-well plates (Nunc) using 100 lL/well and the resorufin fluorescence (kex = 530 nm, kem = 590 nm) was measured in the microplate reader. Untreated cells were taken as control with 100 % viability. All experiments were run in triplicate from two independent measurements. 2.11 In vitro transfection procedure and luciferase activity measurement One day before transfection, HEK 293T cells were seeded onto 24-well tissue culture plates (Corning) at 6 9 104 cells/well, along with 1 mL of DMEM medium containing serum and antibiotics. It should be noted that HEK 293T cells were seeded in the well plates coated with collagen (type I, 0.2 mg/mL in 0.25 % acetic acid). Cells were transfected at 60–70 % confluence. One hour prior to transfection, the cell culture medium in each well was replaced with 0.5 mL fresh serum-free medium. Subsequently, the transfection was performed by adding 100 lL

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(1.5 lg pDNA/well) of the prepared polyplexes to each well. It is worth mentioning that the optimal amount of DNA for transfection varies with different cell lines and media. Nevertheless, the recommended concentration of DNA is generally 1–3 lg/well [18]. The polyplexes were allowed to remain in the cell culture medium for 6 h, after which the cell culture medium was replaced with 1 mL serum-containing medium for an additional 18 h (before cytotoxicity test). Forty eight hours post-transfection, the medium was discarded and 100 lL Reporter Lysis Buffer 19 (RLB) was added to each well and the plates were stored at -80 °C overnight. Cell lysates were analyzed for luciferase activity with Promega’s luciferase assay reagent in triplicate (following the supplier’s instructions). For each sample, the microplate reader was set for 3 s delay with signal integration for 10 s. The amount of protein in cell lysates was determined using the bicinchoninic acid assay (BCA assay) with bovine serum albumin as a standard [19]. The transfection efficiency was characterized by Firefly luciferase expression and denoted as relative light units per mg of protein (RLU/ mg protein) ±standard deviation. All samples were carried out in triplicate and two independent experiments were performed to verify the reproducibility. Non-transfected cells were used as negative controls. 2.12 Fluorescence microscopy Enhanced Green Fluorescent Protein expression studies were carried out 48 h after transfection. An inverted microscope (TE2000-E, Nikon) was used. The NIS Elements Advanced Research software (version 2.31) was utilized to acquire pictures of the transfected cells. 2.13 Computational analysis The interaction between the unmodified/grafted PEIs and DNA was explored using docking calculations. Due to limitations of the program, PEI trimers were used to represent the polymer. Several docking runs were performed, with grafting degrees of [0 %], [25 %], [50 %], [75 %] and [100 %]. The NMR structure of 17-mer DNA duplex (PDB code:1IR5 [20]) was used to represent DNA. Docking was performed with the Vdock program [21]. The solvent effect was modeled with the distance-dependent dielectric approximation, eij = 4rij [22]. The host molecule was kept rigid during docking, while all dihedrals in the guest molecules were rotatable, except for the small CH3 and NH3? groups. Unmodified and grafted PEI trimers were prepared for docking using Avogadro software, v. 1.0.0 and typed with CHARMmÒ force field with Momany–Rone charges [23]. CHARMM27 force field parameters [24] were used for the DNA. The force field parameters

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were generated using Discovery Studio Visualizer v. 3.0 (Accelrys Software Inc., San Diego, CA). A large trans˚ 9 28 A ˚ 9 12 A ˚ ) slicing through the lational box (28 A middle part of DNA was used to avoid biasing of the docking towards any particular site on the surface of the DNA. The number of samplings was increased from the default 3,000–10,000 (15,000 for the 100 % grafted PEI) in the hunt phase of docking, and from 3,000 to 5,000 in the fine tune phase [21]. Instead of the default 20 minima, 100 minima were generated during docking. 2.14 Statistics All statistical analyses were performed using IBM SPSS Statistics 19 software. Results are reported as mean ± standard deviation. One-way ANOVA with Tukey’s Post Hoc test were used to assess the statistical differences between the group means.

3 Results 3.1 Synthesis and characterization of DMAEMA-g-PEI The synthesis of DMAEMA-g–PEI is illustrated in Fig. 1a. The DMAEMA-g–PEI was synthesized by Michael addition reaction between the carbon double bond of DMAEMA and the amino groups of the branched PEI. The grafting degree (GD) on PEI was stoichiometrically controlled by varying the feed amount of DMAEMA in the beginning of the reaction (ranging from 40 to 360 lL), while maintaining the amount of PEI at a fixed value (0.5 g). The PEI and DMAEMA-g–PEI polymers were analyzed by 1H NMR. Representative 1H NMR spectra of the PEI and DMAEMA-g–PEI with GD of [42 %] are shown in Fig. 1b. Compared to PEI which showed typical peaks from d = 2.6–3.6 ppm, a new peak correspondent to the methyl group protons of DMAEMA appeared for PEI-g–DMAEMA (d = 1.1 ppm) [16]. This indicates that DMAEMA was successfully grafted onto PEI. No double bond peak was present in all the modified PEIs, indicating complete removal of the monomers. The GD was determined from the ratio between the areas of the characteristic peaks of PEI (d = 2.6–3.6 ppm) and the peak associated with the methyl protons present in DMAEMA (d = 1.1 ppm). The increase of DMAEMA feeding amount from 40 to 360 lL in the beginning of the reaction resulted in an increased GD of DMAEMA-g-PEI. According to the 1H NMR results, the average number of conjugated DMAEMA monomers increased from 48 to 166 per PEI molecule, resulting in a GD ranging from [25 %] to [88 %], as listed in Table 1.

J Mater Sci: Mater Med (2012) 23:2967–2980 Fig. 1 a Schematic illustration of the synthesis of N,Ndimethylaminoethyl methacrylate grafted poly(ethylenimine) (DMAEMA-g-PEI); b 1H NMR spectra of PEI and DMAEMAg-PEI [42 %] in D2O

(a)

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H2N

PEII

H N

DMAEMA NH2 N

N

N

+

NH2

N

N

NH2

N

O

n

NH2

O

NH2

N

Methanol

Room Temperature N 2 ,72h

O N O

HN

DMAEMA-g-PEI

H N

NH2 N

N

N

NH2

N

N

NH2

n

N

H N

O

N

NH2

O

(b)

Concurrently, the molecular weight (Mw) of the conjugates varied from 32,540 to 51,084 Da.

3.2 Polymer buffering capacity The buffering capacity of unmodified and grafted PEIs was evaluated by acid–base titration. As shown in Fig. 2a, the buffering capacity of the unmodified PEI is only slightly higher than those of DMAEMA-g–PEIs with different grafting degrees. That is, the obtained acid–base titration curves for DMAEMA-g–PEIs with increasing grafting degree are fairly similar to that of the unmodified PEI.

3.3 pDNA condensation In the current study, the extent of pDNA condensation, achieved by the different charged polycations, was evaluated by performing the PicoGreenÒ test. PicoGreenÒ is a high affinity intercalator that is practically non-fluorescent in its free form and become highly fluorescent when bound to free double stranded DNA [25]. Complexes prepared with pDNA and different concentrations of PEI and DMAEMA-g–PEI of various grafting degrees were analyzed by measuring the fluorescence intensity of PicoGreenÒ. As shown in Fig. 2b, the fluorescence intensity decreased with increasing polymer concentration, revealing

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Table 1 Conditions and results of unmodified and grafted PEI PEI (g)

a

DMAEMA (lL)

a

b

GD (NMR) (%)

DMAEMA/ PEIc

48

Mw of modified PEI (Da)

0.5

40.0

25

32,540

0.5

180.0

42

78

37,304

0.5

360.0

88

166

51,084

a

Initial amount used in the reaction

b

Grafting degree of PEI calculated based on NMR information

c

Number of grafted DMAEMA per a PEI based on NMR information

the ability of the vectors to compact the pDNA. In general but more evident at lower polymer concentrations, the grafted PEIs were less tightly bound to pDNA, as indicated by a lower percent reduction in fluorescence intensity than unmodified PEI. The fluorescence curves reached a plateau at polymer concentration of C2.5 lg/mL, suggesting the large condensation of pDNA at or above this concentration, in which pDNA is no longer accessible to intercalating fluorochromes. Figure 2c shows the calculated N/P ratio for given polymer concentrations in the current study. The N/P ratio of the DMAEMA-g-PEI/DNA complexes is defined as the molar relation of primary amine groups in the cationic molecule (secondary and tertiary amines are neglected in this calculations due to their lower pKa values), which represent the positive charges, to phosphate groups in the DNA, which represent the negative charges. The calculation of the N/P ratio was based on the assumption that one repeating unit of PEI featuring one nitrogen corresponds to 43.1 g/mol, and one repeating unit of DNA featuring one phosphate corresponds to 330 g/mol [26]. As presented in Fig. 2c, the N/P ratio increases with increasing the polymer concentration and decreasing the grafting degree. 3.4 Size and zeta potential of the polyplex Polyplexes of unmodified and grafted PEIs were formed at various polymer concentrations and their mean size and zeta potential were investigated (Fig. 2d). In the present study, no significant differences were noticed among the size of the studied polyplexes. All synthesized polymers were able to compact pDNA into nanoparticles with the mean size of around 115 nm. The zeta potential of free pDNA was found to be -52.3 ± 8.8 mV. As seen in Fig. 2d, increasing the polymer concentration resulted in higher zeta potentials. In other words, the negative charge of pDNA is counterbalanced by the positive charge of the polycations. The results also show that grafting DMAEMA monomer onto PEI did

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not lead to significant changes in surface charge of complexes when the same polymer concentration was used. It is worth mentioning that, since the zeta potential measurements were carried out at low ionic strength (pure water), the zeta potential of the particles under the physiological ionic strength, where transfection was observed, will be lower than the zeta potential values shown in Fig. 2d [13]. Thus, the zeta potential data should not be evaluated in absolute terms but rather be used for comparison among the different polymers. 3.5 Cytotoxicity of polymers The cytotoxicity of DMAEMA-g–PEI/pDNA complexes was evaluated in HEK 293T cells using the resazurin reduction assay. The unmodified PEI (25 kDa)/pDNA complex was used as a positive control, and cells incubated without any polymer complex were used as a negative control. The cells were incubated with the polyplexes for 6 h in serum-free medium and the resazurin assay test was performed 24 h post-transfection. It came as no surprise that the complexes formed by the grafted PEIs were less toxic than the unmodified PEI complexes (Fig. 3a). The reduction in toxicity of PEI is expected to be a function of DMAEMA grafting degree. This difference in cell viability was more evident between DMAEMA-g-PEI [88 %]/pDNA and unmodified PEI/ pDNA complexes. For the unmodified PEI/pDNA and low grafted DMAEMA-g-PEI/pDNA complexes signs of growth inhibition were evident at high polymer concentration (20 lg/mL) from the presence of rounded and detached cells on the substrate. On the contrary, cells transfected with lower polymer concentrations and also high grafted DMAEMA-g-PEI/DNA complexes remained healthy and appeared similar to control untreated cells. Also, as was expected, cell viability decreased with increasing polymer concentration. Taking cell viability into consideration, DMAEMA-gPEI [88 %]/DNA complexes are more competent vectors in the tested cell line and at the given concentrations. 3.6 In vitro transfection studies The gene transfection efficiency of DMAEMA-g-PEI/ pDNA complexes was assessed by in vitro delivery experiments of luciferase and GFP reporter genes (pEGFPLuc) into HEK 293T cell line. The results of luciferase activity were normalized for the protein content in culture and are shown in Fig. 3b. The unmodified PEI (25 kDa)/ pDNA complex was used for comparison. The polymers were complexed at three different concentrations (5, 10 and 20 lg/mL) with pDNA (pEGFP-Luc) to form polyplexes in the culture medium. The cells were incubated with the

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Relative fluorescence (%)

(a) 10

pH

8 6 4 2 0

(b) 100

*

60

* *

40

*

20 0

0

0.5

1

1.5

2

2.5

0

3

Titrant Volume (mL) Polymer ConcentratIon (µg/mL)

*

80

0.1

0.25

0.5

1

2.5

5

10

Polymer concentration (µg/mL)

25

(c) 20

PEI

15

PEI-g-DMAEMA [25%] 10

PEI-g-DMAEMA [42%] PEI-g-DMAEMA [88%]

5

0 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

N/P ratio 60

(d) 1250

30 250 20

*

* 10

Zeta potential (mV)

40

Size (nm)

20 µg/mL

50

10 µg/mL 5 µg/mL 20 µg/mL 10 µg/mL 5 µg/mL

0

50 PEI

DMAEMA-g-PEI [25%]

DMAEMA-g-PEI DMAEMA-g-PEI [42%] [88%]

Fig. 2 a Acid–base titration profiles of PEI (branched, 25 kDa), and DMAEMA-g-PEI of various grafting degrees in 150 mM NaCl solution (titrated using 0.1 M NaOH); b PicoGreen (PG) assay. The results are reported as the relative percentage of PG fluorescence, where 100 % intensity was observed for zero concentration of polyplexes (only pDNA). *P \ 0.05 when the grafted PEI complexes are compared with the unmodified PEI complex at the same polymer concentration; c Correlation of the polymer concentration with N/P

ratio. For each polymer tested, the N/P ratio was determined at various concentrations, in particular 5, 10, and 20 lg/mL; d The particle size and zeta potential of DMAEMA-g-PEI/pDNA and PEI/ pDNA complexes at various polymer concentrations. Data are shown as mean ± SD (n = 3). *P \ 0.05 when the grafted PEI complexes are compared with the unmodified PEI complex at the same concentration

polyplexes in serum-free medium for 6 h, and analysed for luciferase activity 48 h post-transfection. At polymer/pDNA concentration of 5 lg/mL, the grafted polymers did not show any significant improvement in transfection efficiency compared to the unmodified PEI/

pDNA complex. This concentration seems to be the optimum in gene delivery experiments for the unmodified PEI/ pDNA complex in HEK 293T cells, as it corresponds to the lowest level of cytotoxicity and to the higher values of transfection efficiency. In HEK 293T cell line and for a

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(a)

*

Cell Viability (%)

100 80 60 40 20 0

cells

PEI

DMAEMA-g-PEI [25%]

DMAEMA-g-PEI [42%]

DMAEMA-g-PEI [88%]

RLU / mg protein ( × 10 6)

3000

(b)

*

Cells

2500

20 µg/mL

2000 1500

10 µg/mL

1000

5 µg/mL 500 0 PEI

DMAEMA-g-PEI

DMAEMA-g-PEI

DMAEMA-g-PEI

[25%]

[42%]

[88%]

Fig. 3 a Cytotoxicity evaluation of DMAEMA-g-PEI/pDNA complexes of various grafting degrees as compared to the free PEI/pDNA complex in HEK 293T using resazurin reduction assay 24 h posttransfection. *P \ 0.05 when the complexes are compared with cells; b In vitro Luc gene expression achieved with DMAEMA-g-PEI/

pDNA complexes of various grafting degrees and free PEI/pDNA complexes 48 h post-transfection in HEK 293T cells. *P \ 0.05 when the grafted PEI complexes are compared with the unmodified PEI complexes at the same concentration

given concentration, increasing grafting degree led to a steadily increasing in gene expression, with the exception of DMAEMA-g-PEI [88 %]/pDNA complex at 5 lg/mL in which a sharp and unexpected decrease in transfection efficiency was observed. Thus, of the three concentrations tested, DMAEMA-g-PEI/pDNA with [88 %] grafting degree appeared to be the favoured complexes at higher concentrations of 10 and 20 lg/mL, in HEK 293T cell lines. To further evaluate the in vitro transfection activity of the complexes, the transfection of DMAEMA-g-PEI/ pDNA and PEI/pDNA complexes at varying concentrations and grafting degrees were studied in HEK 293T cells based on the green fluorescent protein (GFP) expression. Figure 4 shows fluorescence and bright field micrographs of the DMAEMA-g-PEI [88 %]/pDNA complexes at three different concentrations in comparison with the unmodified PEI/pDNA complexes. Fluorescence micrographs were then layered over bright field micrographs and merged together. The resultant fluorescence images were in

agreement with the luciferase reporter assay data, in which the number of transfected HEK 293T cells using DMAEMA-g-PEI [88 %]/pDNA complexes is more than PEI/ pDNA complexes at high concentrations of 10 and 20 lg/ mL. The corresponding bright-field images indicated that less cells were adherent to the substrate at higher polymer concentration (20 lg/mL) and in particular for PEI/pDNA complex. This observation is also in line with the high cytotoxicity of the complexes at this concentration.

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3.7 Docking studies The aim of the docking studies was to gain insight into the DMAEMA-g-PEI/pDNA binding modes and energies, and to further understand the higher transfection efficiency obtained with DMAEMA-g-PEI [88 %]/pDNA than with the PEI/pDNA polyplex at the higher polymer concentrations. Vdock docking program [21] has previously been shown to be capable of providing useful benchmarks when a large number of rotatable torsional angles were present during the

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5 µg/mL

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

(o)

(p)

(q)

(r)

PEI/pDNA

DMAEMA-g-PEI [88%]/pDNA

20 µg/mL

Fig. 4 Fluorescence microscopy images (GFP expression) and corresponding bright field and merge micrographs of DMAEMA-g-PEI [88 %]/ pDNA complexes (a–i), and PEI/pDNA complexes (j–r) at concentrations of 5, 10, and 20 lg/mL in HEK 293T cells

calculation [27]. Due to the natural limitations of the docking program, trimers of DMAEMA-g-PEI were used for simulations instead of the polymers. Solution NMR structure of 17-mer DNA was utilized as a rigid receptor [20]. Because of the system size and the feature richness of DNA and DMAEMA-g-PEI molecules (i.e. multiple

charged atoms and a complex receptor surface), multiple binding modes were found with closely spaced binding energies. For this reason, 5 best binding modes were analyzed for each ligand/receptor pair (Fig. 5a–e). The top hits exhibited considerable overlap, signifying qualitative trends.

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Fig. 5 Five best hits of unmodified and grafted PEI trimers (colored sticks) docked to DNA, with grafting degrees of: a [0 %], b [25 %], c 75 [50 %], d [75 %], e, f [100 %]. The complex is viewed from the opposite side of the DNA for (a–e) to better discern the position of the

docked trimers with respect to the minor and major grooves of DNA. A docked top hit for DMAEMA-g-PEI [100 %] (green sticks) is additionally shown in (f), viewed from the ‘‘front’’ to highlight interactions with DNA

Figure 5a–e shows significant change of the trimer position with respect to the minor groove of DNA as the grafting degree increases. The unmodified PEI binds exclusively to the minor groove of the DNA (Fig. 5a). This presumably is due to the narrowness and the high positive charge of the unmodified PEI, and the high negative charge of the relatively narrow minor groove of DNA. Grafted PEIs with [25 %] and [50 %] grafting degrees also bind in the minor groove (Fig. 5b, c); however, a closer inspection reveals that the docked trimers exhibit a somewhat less perfect fit to the minor groove. Due to the length of docked trimers, DMAEMA substituted ‘‘side branches’’ of PEI show a tendency to dangle outside of the minor groove. By increasing the grafting degree to [75 %], the DMAEMA-g– PEI trimer only in the middle part binds inside of the minor groove, and the rest of the trimer dangles outside of the minor groove making some contacts with the phosphates of the next coil by bridging across the major groove, without dipping deeply into it (Fig. 5d). For the fully grafted

[100 %] DMAEMA-g-PEI trimer, the angle between the polymer backbone and the minor groove becomes nearly perpendicular (Fig. 5e). Figure 5f represents the top hit of DMAEMA-g-PEI [100 %] from Fig 5e. Two of DMAEMA-containing ‘‘side branches’’ of DMAEMA-g-PEI extend perpendicularly from the polymer backbone, binding in the minor groove of DNA. The rest of the trimer bridges the major groove, making contacts with the phosphates in the next phosphate-deoxyribose coil of DNA. The interaction energy reported by docking between the ligand and receptor decreases in magnitude with increasing grafting degree, despite the increase of the ligand size (e.g. for DMAEMA-g-PEI [25 %]: -264 kcal/mol; [50 %]: -253 kcal/mol; [75 %]: -246 kcal/mol; and [100 %]: -207 kcal/mol). The resultant decrease in interaction energy is consistent with the observed increase in number of dangling ‘‘side branches’’ of DMAEMA-g-PEI and worsened fit against the minor groove of the DNA. Full length [0 %], [25 %], and [50 %] grafted polymers are

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predicted to bind in the minor groove along their entire length. For the grafting degrees of [75 %] and [100 %], due to the limited sizes of the DMAEMA-g-PEI trimer and the DNA receptor used for the docking, it is difficult to predict in detail how the full length polymer and DNA would bind. However, the calculations show a marked change in the binding mode as compared to binding of the low grafting degree polymer.

4 Discussion 4.1 Buffering capacity and DNA condensation of DMAEMA-g-PEI Polymer buffering capacity can have a significant effect on gene expression in mammalian cells. Once polyplexes enter cells by endocytosis, and after fusion of the endosomes with lysosomes via forming endolysosomes, the polymer buffering capacity can protect the DNA from the action of nucleases that work at low pH values. Furthermore, it can facilitate endosomal escape through the proton sponge effect [28]. The polymer buffering capacity leads to protons pumping and concomitant influx of chloride anions into the endosomes, thereby increasing their internal ionic strength and causing their osmotic rupture. Consequently, pDNA is released to the cytosol and endolysosomal trafficking of pDNA is circumvented. Optimal buffering capacity at endosomal pH is essential to further enhance endosomal escape of polyplexes into the cell cytoplasm [29]. The present work shows similar trends in acid–base titration curves for DMAEMA-g-PEIs and for PEI. The DMAEMA grafting onto PEI only slightly decreases its buffering capacity. The buffering capacity of the polycations mainly depends on the presence of primary, secondary and tertiary amine groups which possess different pKa values (around 9, 8 and 6–7 for primary, secondary and tertiary amines, respectively) [30]. Having in mind that branched PEI has 25 % primary, 50 % secondary, and 25 % tertiary amine groups [31], the relatively lower buffering capacity of the grafted PEIs can be due to the lower density of primary amines in their structure and higher density of secondary and tertiary amines. These observations are in line with our expectation. Another important factor in cell transfection is the condensation of DNA that enables the DNA to be transported into the target cells. Condensation of DNA, as defined by Bloomfield [32], is a reaction that occurs spontaneously when DNA phosphate charge is 90 % neutralized. This process can also be defined as a decrease in DNA volume upon transition from the extended chain to a compacted state. Condensation protects DNA from

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degradation by nucleases, and the compact particles can be taken up by cells via natural processes. The obtained results of PicoGreenÒ analysis (as shown in Fig. 2b) indicated that all the DMAEMA-g-PEI polycations prepared in this study were able to efficiently bind and condense pDNA at or above a certain concentration (C2.5 lg/mL). These results were plausible, since both PEI and pDMAEMA have been shown to be capable of condensing pDNA into positively charged polyplexes that bind and transfect cells [14, 33]. The primary amine based polymers show better DNA condensation ability and DNA protection [34]. Therefore, it is clear that the larger number of primary amines (high charge) on the unmodified PEI helped it to bind electrostatically to the polyanionic pDNA, whereas the reduced number of primary amines (low charge) of the grafted PEIs resulted in weaker interactions with pDNA. However, it should be noted that the relatively weaker condensation capability of the grafted PEIs can facilitate release of pDNA from the complexes after entering cells, and thereby enhance the transfection efficiency. 4.2 Size and surface charge of DMAEMA-g-PEI The degree of cellular uptake and transfection efficiency strongly depends on the particle size and surface charge of polycation/DNA complexes. pDNA has a considerable large hydrodynamic size and does not readily penetrate most cellular membranes unless complexed with cationic polymers (e.g. unmodified and grafted PEIs). These positively charged polymers are capable of condensing the extended structure of plasmids and form small nanoparticles. In the current study, the grafted PEI complexes with the mean size of around 115 nm (Fig. 2d) are in a suitable range for an efficient entry into the cells. Win et al. [35] demonstrated that nanoparticles of 100–200 nm size acquire the best properties for cellular uptake, while smaller (50 nm) or larger particle size (500 and 1,000 nm) resulted in lower cellular uptake.van de Wetering et al. and Cherng et al. [11, 13] also reported that the highest transfection efficiency was found for polyplexes with a mean size around 150 nm and a positive zeta potential (30–45 mV). Although the optimal size for cellular internalization may be dependent on the characteristics of the target cells [36], the obtained range of particle size in the current study should allow the nanoparticles to enter the cell preferably through the process of endocytosis. In size range of approximately 20–200 nm (diameter), particles are taken up through endocytosis. Above this size phagocytosis is thought to predominate [37, 38]. However, even carriers as large as 500 nm have been known to be internalized via receptor-mediated endocytosis [39].

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Besides particle size, the zeta potential of polymerbased complexes is a key parameter in determining transfection efficiency. As seen in Fig. 2d, increasing the polymer concentration led to higher zeta potentials. From a practical point of view, this gives rise to solutions with a better colloid stability and easier handling. The overall net positive charge of the unmodified and grafted PEI complexes in the current study is indicative of high pDNA condensation capabilities of the polycations which is considered to be advantageous for gene delivery. It enhances the electrostatic interaction of the cationic complex with the negatively charged cell membrane (by promoting its uptake into target cell), protects the pDNA once inside the endolysosomal compartment, and facilitates the complex endolysosomal escape [34, 40]. However, the high charge density is also another factor that may contribute to the cytotoxicity of a cationic complex [41]. It thus becomes imperative to strike a balance between the pDNA condensation and charge density for an efficient gene delivery system. 4.3 Cytotoxicity and gene transfection An ideal gene delivery vector should be able to mediate sufficient levels of gene transfection without compromising the viability of the host cells. However, the level of cytotoxicity varies with cell lines and the nature of the vector (e.g. polycation) [42]. Our results for HEK 293T cells show that the pDNA complexes with the grafted PEIs were less toxic than with native PEI. Indeed, it is known that polycationic vectors such as PEI cause extensive levels of toxicity, which is thought to be a result of membrane damaging effects [43]. The published mechanisms of cytotoxicity mediated by PEI, and to some extent pDMAEMA, indicated a typical feature of necrotic cell death [42, 44, 45]. The necrosis-like cell death is often much faster to take place, whereas apoptosis-like cell death takes longer and in some cell systems it may take days [42]. In the current study the fast rate of cell degeneration at higher polymer concentrations (esp. for PEI polyplexes) and at relatively short incubation time of the polyplexes (6 h), can be associated to necrotic cell death. Furthermore, the reduction in cell viability with increasing polymer concentration is associated with high charge density of the system and possibly higher amount of free cationic polymers leading to higher cytotoxicity [46]. In HEK 293T cell line and for a given polymer concentration, increasing grafting degree led to a steadily increasing in gene expression. The obtained results of DMAEMA-g-PEI with higher grafting degree are comparable to the PEI grafted with polyglycerol and chitosan in similar conditions, as reported by Zhang et al. [47] and Wong et al. [28], respectively. The size and surface charge

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of polyplexes may have an important impact on overall transfection efficiencies. In the current study, due to the similar range of particle sizes at all concentrations and polymer grafting degrees, the difference in transfection efficiency cannot be explained based on the particle size. Moreover, for a specific polymer concentration, the surface charge of polyplexes is also similar and cannot be used as a justification for the obtained results. As such, computational analysis was used in an attempt to understand the higher transfection efficiencies obtained with DMAEMAg-PEI/pDNA than with the PEI/pDNA polyplexes. The docking studies clearly indicate that the binding modes of the highly grafted DMAEMA-g-PEI to DNA significantly differ from the low grafted PEI variants, and that the interaction energy between the polymer and DNA decreases in magnitude with increasing grafting degree. The conducted qualitative study is generally consistent with our gene transfection results, allowing us to state that the higher transfection efficiencies achieved by the DMAEMA-g-PEI [88 %]/pDNA polyplex can be associated with the weaker interactions established between the polymer and DNA. The weak interactions give rise to an easier unpacking and separation of the nucleic acid from the vector after cellular uptake. These findings are in agreement with the previous PicoGreenÒ results, which showed that the grafted PEIs were less tightly bound to pDNA when the same polymer concentration was used.

5 Conclusions In this study, the effect of DMAEMA conjugation onto PEI was studied. The DMAEMA-g-PEI conjugates of variable grafting degrees was successfully synthesized, characterized and evaluated as a gene carrier in HEK 293T cell line. Our results showed that the DMAEMA grafted polymers with higher grafting degree had lower cytotoxicity than PEI alone, in particular at high polymer concentrations. DMAEMA-g-PEI retained the good binding ability and buffering capacity of PEI and showed enough ability to condense pDNA into nanoparticles with proper size and zeta potential to be internalized by cells. Furthermore, at high concentrations, the DMAEMA-g-PEI [88 %] was found to be the most efficient gene delivery vector. Computational analysis using docking calculations suggested the possibility of establishing a correlation between the gene delivery results and the decrease in interaction between the vector and DNA with increasing grafting degree. That is, pDNA will easily unpack and release from the carrier once inside the cell at higher grafting degree. Our results indicate that the grafting of DMAEMA onto PEI is a promising approach to improve its gene delivery performance.

J Mater Sci: Mater Med (2012) 23:2967–2980 Acknowledgments The Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT, Portugal) is acknowledged for funding through the project PEst-OE/QUI/UI0674/2011 (CQM, Portuguese Government funds) and the NMR Portuguese Network (PTNMRREDE/1517/RMN/2005POCI2010/FEDER). The support of FCT through the scholarship SFRH/BPD/47369/2008 (A. Nouri) and the Science 2008 program (Y. Li) is also acknowledged.

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