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Influence of hydroxyl groups on the biological properties of cationic polymethacrylates as gene vectors
Acta Biomaterialia 6 (2010) 2658–2665
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Influence of hydroxyl groups on the biological properties of cationic polymethacrylates as gene vectors Ming Ma, Feng Li *, Zhe-fan Yuan, Ren-xi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education, College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, People’s Republic of China
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Article history: Received 4 August 2009 Received in revised form 10 January 2010 Accepted 15 January 2010 Available online 22 January 2010 Keywords: Cationic polymers Gene delivery Hydroxyl Atom transfer radical polymerization
a b s t r a c t In this study poly(aminoethyl methacrylate) (PAEMA), poly(3-amino-2-hydroxypropyl methacrylate) (PAHPMA), poly(2-(2-aminoethylamino)ethyl methacrylate) (PAEAEMA) and poly(3-(2-aminoethylamino) 2-hydroxypropyl methacrylate) (PAEAHPMA) were synthesized using atom transfer radical polymerization to evaluate the effect of hydroxyl groups on the relative properties of cationic polymeric gene vectors. The results of heparin displacement assays showed that PAHPMA possessed a stronger binding capacity than PAEMA. PAHPMA/DNA complexes and PAEAHPMA/DNA complexes had lower zeta potentials than those of PAEMA and PAEAEMA. MTT assay results indicated that PAHPMA and PAEAHPMA exhibited obviously lower cytotoxicities than PAEMA and PAEAEMA. Subsequently, in vitro gene transfection studies in 293T cells without serum showed that PAHPMA exhibited a lower transfection efficiency than PAEMA and PAEAHPMA/DNA complexes possessed a similar transfection efficiency to PAEAEMA/ DNA complexes. Moreover, PAHPMA and PAEAHPMA retained similar transfection efficiencies in DMEM with 10% serum, but PAEMA and PAEAEMA showed slightly lower transfection efficiencies than in the absence of serum. The reason for these phenomena might be attributed to the introduction of hydroxyl groups into PAHPMA and PAEAHPMA, i.e. the existence of hydroxyl groups might increase the binding capacity to DNA and at the same time decrease the surface charge of the polymer/DNA complexes due to the formation of hydrogen bonds between the polymers and DNA. Therefore, a lower zeta potential and stronger binding ability may result in a lower gene transfection efficiency. This effect of hydroxyl groups decreased with increasing amino group density on the polymer. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Non-viral vectors have attracted more and more attention due to their low immune response and low antigenicity, as well as simplicity of preparation [1–3]. Cationic polymers, such as polyethyleneimines (PEIs), poly(dimethylamino ethyl methacrylate) (PDMAEMA), poly(L-lysine) (PLL), and polyamidoamines [4–9], are the major type investigated in the past decades. However, most polycation vectors suffer from unsatisfactory transfection efficiencies and have remained at the laboratory stage until now. Ideal non-viral vectors should be non-toxic, form compact DNA particles of virus-like dimensions, protect the DNA from degradation and facilitate targeted cell binding and internalization, endosomal escape, trafficking the cytoplasmic environment and localizing into the nucleus, as well as vector unpacking [10–15]. In reality, no polycation is able to carry out all the extracellular and intracellular delivery functions. To improve the transfection efficiency of polycation vectors additional functional elements, e.g. poly(ethylene glycol), cyclodextrins, cholesterol, L-arginine and/or hydroxyl groups, need * Corresponding author. Tel./fax: +86 27 68754509. E-mail addresses: [email protected], [email protected] (F. Li).
to be incorporated to modify the polymers such that they exhibit different biological characteristics from unmodified ones. This indicates that subtle differences in chemical structure can have a significant impact on the process of transfection [16–21]. For example, Reineke detailed the effect of hydroxyl groups on the process of gene delivery based on a new library of poly(glycoamidoamine)s synthesized by condensation polymerization of esterified D-glucaric acid, dimethyl meso-galactarate, D-mannaro-1,4:6,3-dilactone and dimethyl L-tartarate with oligoamines. The results showed that the poly(glycoamidoamine) structures containing four secondary amines between the carbohydrate repeat units delivered plasmid DNA containing the firefly luciferase reporter gene with high efficacy into a variety of mammalian cell lines (BHK-21, HeLa and HepG2 cells) in a non-toxic manner, and the biological properties, particularly the delivery efficiency, of these polymers were affected by the hydroxyl groups and their stereo structure together [22–24]. Until now many groups have reported that the inclusion of hydroxyl groups in polycations has helped reduce cytotoxicity and improved gene delivery efficiency. For example, Kang found that incorporating poly(2-hydroxyethyl methacrylate) into poly(dimethylaminoethyl methacrylate) block polymers could enhance transfection efficiency due to the good biocompatibility and shielding effect of the
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.01.024
M. Ma et al. / Acta Biomaterialia 6 (2010) 2658–2665
poly(2-hydroxyethyl methacrylate) blocks [25]. However, Tziveleka indicated that a fourth generation polypropyleneimine dendrimer lost its transfetion efficiency when the primary amino groups in the dendrimer were completely replaced with hydroxylated moieties [26]. Thus, a better understanding of such structure–bioactivity relationships is required. Here we synthesized four polymers, poly(aminoethyl methacrylate) (PAEMA), poly(3-amino-2-hydroxypropyl methacrylate) (PAHPMA), poly(2-(2-aminoethylamino) ethyl methacrylate) (PAEAEMA) and poly(3-(2-aminoethylamino) 2-hydroxypropyl methacrylate) (PAEAHPMA), with similar chemical structures to evaluate their differences in a number of parameters, such as binding capacity and gene delivery. This study will be helpful in further elucidating the effect of hydroxyl groups on the biological properties of polymeric vectors.
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2.4. Synthesis of PAEAHPMA PGMA (0.1 g) and Boc-EDA (2.3 g) were dissolved in 50 ml of DMF and reacted at 70 °C for 4 h. After the reaction the mixture was added to 5 M HCl and stirred for 5 h, and then dialyzed against excess HCl solution (2 l, pH 3) with three changes of acid a day for 2 days. The polymer was obtained by freeze drying. 2.5. Synthesis of PAEMA and PAEAEMA The Boc-protected PAEMA and Boc-PAEAEMA were prepared by a similar approach to the synthesis of PGMA at the same initiator/ monomer ratio (1:70). The resultant polymer was stirred in 5 M HCl for 3–5 h. After the signal for tert-butoxycarbonyl groups completely disappeared from 1H NMR spectra the solution was dialyzed for 2 days. PAEMA and PAEAEMA were collected after freeze drying.
2. Materials and methods 2.1. Materials
2.6. Polymer characterizations 1
Glycidyl methacrylate (GMA) (Aldrich, 97% pure) was purified by vacuum distillation under reduced pressure and stored in a refrigerator under a nitrogen atmosphere. N-(2-hydroxyethyl)ethlyenediamine was purchased from Alfa. Copper (I) bromide (Beijing Chemical Co.) was purified by washing with acetic acid and ethanol in turn. Ammonia (27%), methacryloyl chloride, ethanolamine, ethylenediamine, 2,2-bipyridine (Bpy), N,N-dimethylformamide (DMF), dioxane, dichloromethane and ether were all purchased from Beijing Chemical Co. and used without further purification. N-tert-butoxycarbonyl-ethylenediamine (Boc-EDA) and N-tert-butoxycarbonyl-aminoethyl methacrylate (Boc-AEMA) were prepared according to Geall et al. [27] and Dubruel et al. [28]. N,N0 -di-(tert-butoxycarbonyl)-2-(2-aminoethylamino)ethyl methacrylate (Boc-AEAEMA) was synthesized via a similar method to Boc-AEMA. Ethyl 2-bromoisobutyrate (EBIB) (98% pure), Dubelcco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, trypsin, phosphate-buffered saline (PBS) and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich. The reporter plasmid pGL3-Luc was purchased from Promega. The plasmid DNA was stored at 20 °C prior to use.
H nuclear magnetic resonance (NMR) spectrometry (Mercury VX-300) and Fourier transform infrared (FT-IR) spectrometry (AVATAR 360) were used to determine the chemical structures of the synthesized polymers in either CDCl3 or deuterated water, while the molecular weights and molecular weight distributions were estimated by gel permeation chromatography (GPC) using a Waters 2690 separation module and a Waters 2410 refractive index detector, with HAc–NaAc buffer solution (0.03 mol l1, pH 3) as eluent and poly(ethylene glycol) with a narrow molecular distribution as the standard. Transmission electron microscopy (TEM) experiments were performed in a JEM-2010 instrument operating at an acceleration voltage of 200 keV. 2.7. Cell culture 293T cells were incubated in DMEM supplemented with 10 vol.% fetal bovine serum (FBS) and antibiotics (10,000 U ml1 penicillin–streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were subcultured prior to confluence using trypsin–EDTA. 2.8. DNA binding
2.2. Synthesis of poly(glycidyl methacrylate) (PGMA) GMA (3.5 g, 24 mmol), EBIB (0.068 g, 0.352 mmol) and Bpy (0.11 g, 0.7 mmol) were dissolved in 4 ml of DMF in a brown reaction flask and degassed by three freeze–pump–thaw cycles, then CuBr (0.05 g, 0.352 mmol) was added. After another three freeze– pump–thaw cycles polymerization was started under an Ar atmosphere at 60 °C for 7 h. After that the solution was poured into excess ether to precipitate the polymer. The precipitate was dissolved in dichloromethane and purified using an alumina column to eliminate the used copper, then precipitated again in ether. The polymer obtained was dried in vacuum.
2.3. Synthesis of PAHPMA PGMA (0.1 g) was dissolved in dioxane and ammonia (2:1 vol.%) mixed solution in a sealed reactor and reacted at 30 °C for 7 days. Subsequently the mixture was concentrated and dialyzed against excess HCl solution (2 l, pH 3) with three changes of acid a day for 2 days. The polymer was obtained by freeze drying.
The DNA condensation ability of the polymers was assessed by means of an agarose gel retardation assay. Polycation/pDNA complexes with different N/P ratios from 0 to 7 were formed by adding an appropriate volume of polymer solution (150 mM NaCl) to 0.1 lg of pDNA (0.1 mg ml1 in 40 mM Tris–HCl buffer). Then 6 ll of sample containing 1 ll of ethidium bromide and 0.1 lg of DNA were electrophoresed through a 1% agarose gel using Tris–borate–EDTA buffer (pH 7.2) at 80 mV for 80 min. DNA retardation was observed by irradiation with UV light and assayed using Cam2com software. 2.9. Particle size and zeta-potential measurements The particle size and zeta potential were determiner in a NanoZSZEN3600 (Malvern) instrument at room temperature. The polycation/pDNA complexes were prepared according to the previous procedure at N/P ratios ranging from 10 to 50. Then the polyplexes were incubated at room temperature for 30 min. After that the polyplexes were diluted with 150 mM NaCl solution or pure water to 1.0 ml volume for size and zeta-potential measurements, respectively.
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2.10. Heparin displacement The binding intensity of polyplexes was determined by heparin displacement experiments. The sample/DNA complexes were first prepared at an N/P ratio of 20 for 30 min as described above and then incubated with heparin solutions of different concentration (ranging from 0.1 to 2 mg ml1). An agarose gel retardation assay was used to detect the released DNA. 2.11. Cytotoxicity assay The cytotoxicity assay was carried out on the basis of an MTT assay in 293T cells. Cells were seeded in 96-well plates at an initial density of 6000 cells well1 and cultured for 24 h in 200 ll of DMEM. 24 h after polymer was added the medium was replaced with MTT reagent (20 ll in PBS at 5 mg ml1) and then incubated for another 4 h. Thereafter MTT was removed and 200 ll of DMSO was added until the crystals dissolved. The absorbance at 570 nm of the solution in each well was recorded using a Microplate Reader (Bio-Rad model 550). Cell viability was calculated according to the equation: cell viability (%) = (ODsample ODblank)/(ODcontrol ODblank) 100, where ODsample is the absorbance of the solution of cells cultured with the polymer or PEI, ODblank is the absorbance of the medium and ODcontrol is the absorbance of the solution of cells cultured with medium only. 2.12. In vitro transfection For transfection experiments pGL3 plasmid DNA was used to evaluate the transfection efficiency of the four polymers in 293T cells. 293T cells were seeded in 24-well plates at a density of 6 104 cells well1 and cultured with 1 ml of DMEM containing 10% FBS for 1 day at 37 °C in a humidified atmosphere of 95% air and 5% CO2 until the cells reached 70% confluence. The polymer/DNA complexes were formed at different N/P ratios ranging from 20 to 40 according to the conditions described above (containing 1 lg DNA for each unit N/P ratio). The complexes were added to each well and incubated in 1 ml of DMEM at 37 °C for 4 h, then the medium was replaced with 1 ml of DMEM containing 10% FBS and the cells were further incubated for 48 h. After incubation the cells were permeabilized with 200 ll well1 cell lysis buffer. Luciferase activity was measured by detecting the light emission from an aliquot of cell lysate incubated with 100 ll of luciferin substrate (Promega) in a luminometer (Lumat LB9507, Berthold). The light units measured using protein assay kits (Pierce) were normalized against the protein concentration in the cell extracts. Luciferase activity was expressed as relative light units (RLU mg protein1). The procedures for transfection experiments in serum were as described above except that the medium was replaced by DMEM supplemented with 10% FBS.
The 1H NMR spectrum of PGMA is shown in Fig. 1. The peaks at 0.93–1.09 and 1.92–1.97 p.p.m. were ascribed to the methyl and methylene protons on the backbone of PGMA. The peaks at 2.64, 2.84 and 3.23 p.p.m. are characteristic of the epoxy groups on the sidechain of PGMA, and the peaks at 3.79 and 4.42 p.p.m. were assigned to the methylene protons adjacent to the epoxy group for space isomerization of the epoxy group. After nucleophilic reaction of the amines with epoxy groups of PGMA the signal for the epoxy groups completely disappeared and the peak of the methylene protons adjacent to the epoxy groups shifted to 3.92–4.20 p.p.m. for PAHPMA and 3.72–3.99 p.p.m. for PAEAHPMA. The signal for methine connected to hydroxyls also occurred here. The peaks at 2.91–3.50 p.p.m. for PAHPMA and 2.68–3.29 p.p.m. for PAEAHPMA belonged to the characteristic peaks of methylene protons adjacent to amino groups. The ring-opening reaction of epoxy groups was further confirmed by FT-IR (Fig. 2). The characteristic absorptions of the epoxide ring at 849 and 907 cm1 also disappeared after the ring-opening reaction and a broad band at 3431 cm–1, assigned to N–H stretching and O–H stretching, appeared. PAEMA and PAEAEMA possessed similar chemical shift peaks to PAHPMA and PAEAHPMA, except that of methylene protons adjacent to hydroxyl groups. Moreover, disappearance of the peak at 1.41 p.p.m. in the 1 H NMR spectra indicated the entire elimination of protected groups (data not shown). The molecular weights and molecular weight distributions of all polymers were also evaluated by GPC using HAc–NaAc buffer solution as the eluent. According to the results of GPC, the number average molecular weights of PAHPMA, PAEAHPMA, PAEMA and PAEAEMA were 8600, 17,000, 10,000 and 14,500 g mol1, respectively. Thus, the repeating units of these four polymers were 62, 85, 76 and 80, respectively, according to the GPC results. And their molecular weight distributions were 1.99, 2.4, 1.95 and 1.33. The above results indicate that the desired polymers were successfully prepared. 3.2. Gel retardation assay The ability of polymers to condense DNA was first investigated by gel retardation assay. The amount of polymer added was calculated on the basis of the chosen N/P ratio, i.e. polymer nitrogen atoms to DNA phosphorus atoms. As shown in Fig. 3, all polymers exhibited good DNA binding ability. PAHPMA and PAEMA retarded bulk DNA movement at a similar N/P ratio. It seems that there was no obvious difference in the N/P ratio needed when hydroxylated polymers were used. Furthermore, PAEAHPMA and PAEAEMA showed a similar result. However, according to the earlier literature increasing numbers of hydroxyl groups could hinder interaction with DNA due to the neutral functionality [23,31]. Thus, additional experiments to further evaluate the effect of hydroxyl groups on DNA binding will be carried out by heparin displacement assay below. 3.3. Particle size and zeta-potential measurements
3. Results and discussion 3.1. Synthesis of polymers Atom transfer radical polymerization (ATRP), one of the most actively developing areas in polymer chemistry, is a powerful tool for the preparation of well-defined polymers with predetermined molecular weights and molecular weight distributions [29,30]. Thus, in order to synthesize PAHPMA, PAEAHPMA, PAEMA and PAEAEMA with similar degrees of polymerization, ATRP was used to polymerize GMA, Boc-AEMA and Boc-AEAEMA at the same initiator/monomer ratio (1:70). 1H NMR, FT-IR and GPC were used to characterize these polymers (Scheme 1).
The particle sizes of polyplexes were investigated in 150 mM NaCl solution by dynamic light scattering (DLS). As revealed in Fig. 4, all polycations could condense DNA into nanoparticles to be endocytosed (ranging from 157 ± 10 to 381 ± 10 nm). The PAHPMA/DNA and PAEMA/DNA complexes showed a trend of decreasing average diameter with increasing N/P ratio from 10 to 50. No distinct difference in size was observed at parallel N/P ratios for the two polycations/DNA complexes. A similar trend was also observed for the PAEAHPMA/DNA and PAEAEMA/DNA complexes, except that the average size of the PAEAHPMA/DNA complexes was smaller than that of the PAEAEMA/DNA complexes at the same N/P ratio in the range 10–30. It seems that hydroxyl groups on
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Scheme 1. Schematic illustration of PAHPMA, PAEMA, PAEAHPMA and PAEAEMA.
Fig. 1. 1H NMR spectra of PGMA (in CDCl3), PAHPMA and PAEAHPMA (in D2O).
Fig. 2. FT-IR spectra of (a) PGMA, (b) PAHPMA and (c) PAEAHPMA.
PAHPMA and PAEAHPMA had no obvious effect on the size of the polyplexes.
TEM was then used to characterize the morphology of the PAHPMA/DNA and PAEMA/DNA complexes at a N/P ratio of 20. As shown in Fig. 5, both polycations condensed DNA into nanoparticles with a diameter of 300 ± 30 nm. There was no significant difference in size, as in the DLS results. However, the particle sizes measured by TEM were appreciably smaller than those obtained by DLS. This might be because the particle sizes measured by DLS were obtained in the hydrated state in solution, while those obtained by TEM had been dried after dropping polyplexe solution onto carbon-coated copper meshes. The zeta potentials of the polyplexes were measured in pure water, and are shown in Fig. 6. It is obvious that the PAHPMA/ DNA and PAEAHPMA/DNA complexes showed much lower zeta potentials than those of PAEMA and PAEAEMA, respectively. This effect is not as yet clearly understood; we presume that this may be ascribed to the stronger intermolecular hydrogen bonding between the polymers with hydroxyl groups and DNA, which resulted in lower zeta potential values [22]. This is not the same as the case of poly(ethylene glycol) (PEG), because PEG mainly reduces the zeta potential of polyplexes by steric hindrance [16]. The zeta potential of PAHPMA/DNA complexes increased from 3.6 ± 0.2 to 8.6 ± 1.2 mV on increasing the N/P ratio from 10 to
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Fig. 4. Particle sizes of (a) PAHPMA/DNA complexes, (b) PAEMA/pDNA complexes, (c) PAEAHPMA/DNA complexes and (d) PAEAEMA/DNA complexes measured by DLS in 150 mM NaCl solution.
were obviously disrupted at a heparin concentration of 1 mg ml1. This indicates that the hydroxyl groups on the sidechains of PAHPMA might facilitate DNA binding. Furthermore, the zeta potential of PAHPMA/DNA complexes at N/P = 20 was much lower than that of PAEMA/DNA complexes (4.6 ± 0.2 vs. 13.4 ± 1.2 mV), which suggests that the lower surface charge of the PAHPMA/DNA complexes might be less attractive to negatively charged heparin, resulting in less replacement of DNA. This may be another explanation for the above heparin displacement results. The PAEAHPMA/DNA complexes exhibited obvious disassembly at 1 mg ml–1 heparin, like PAEAEMA, but disassociation was weaker than that of PAEAEMA/ DNA complexes at the same heparin concentration. This might indicate that PAEAHPMA possesses a slightly stronger binding ability than PAEAEMA on the one hand, while, on the other hand, the shielding effect of hydroxyl groups decreases, in comparison with PAHPMA, with the introduction of secondary amino groups into PAEAHPMA. The above results indicate that the presence of hydroxyl groups in PAHPMA and PAEAHPMA facilitate DNA binding due to the possible formation of hydrogen bonds between hydroxyl groups on the polymers and DNA base pairs [32]. However, the detailed mechanism of binding and the shielding effect of hydroxyl groups is not yet understood and requires further study. Fig. 3. DNA binding ability of polymers. Electrophoretic mobility of plasmid DNA in (a) PAHPMA, (b) PAEMA, (c) PAEAHPMA and (d) PAEAEMA/DNA complexes at the N/P ratios specified (0–7).
50, while PAEAHPMA/DNA maintained an almost steady zeta potential around 30 mV. The sharp difference in zeta potentials between them might be due to the higher amino group density for PAEAHPMA, which contains additional secondary amino groups. Thus, these results might show that the existence of hydroxyl groups can shield the surface charge of complexes due to the formation of hydrogen bonds and that the shielding effect of hydroxyl groups decreases with increasing amino group density of the polymer. 3.4. Heparin displacement assay To further investigate the binding effect of hydroxyl groups, a heparin displacement assay was carried out, with the N/P ratio for all the polycation/DNA complexes being kept at 20. As revealed in Fig. 7, PAHPMA/DNA complexes remained almost stable up to a heparin concentration of 2 mg ml1, while PAEMA/DNA complexes
3.5. Cytotoxicity of the polymers The cytotoxicity of the four polymers was evaluated in 293T cells by MTT assay. As shown in Fig. 8, PAHPMA and PAEAHPMA exhibited obviously higher relative cell viability than PAEMA and PAEAEMA, and PAHPMA showed 55.45% relative cell viability even when the polymer concentration was 2.75 mg ml1. One probable reason for the lower cytotoxicity of PAHPMA and PAEAHPMA might be their hydroxyl groups, which enhance the biocompatibility of polycations [22]. 3.6. In vitro transfection Gene transfection efficiency was influenced by many factors, such as binding capacity and biocompatibility of the polymers, surface charge and the size of the corresponding polymer/DNA complexes. The above results show that hydroxyl groups on the sidechains of PAHPMA and PAEAHPMA have a significant impact on their properties. These changes may induce different gene transfection efficiencies. Here, 293T cells were used to evaluate the effect of hydroxyl groups on gene transfection activity. Branched 25 kDa PEI, which is the most widely used polycation
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Fig. 5. TEM images of (a) PAHPMA/DNA complexes (b) PAEMA/DNA complexes at N/P = 20.
Fig. 6. Zeta potentials of (a) PAHPMA/DNA complexes, (b) PAEMA/pDNA complexes, (c) PAEAHPMA/DNA complexes and (d) PAEAEMA/DNA complexes at various N/P ratios.
Fig. 8. Relative cell viability of 293T cells co-incubated with polymers for 24 h: (a) PAHPMA; (b) PAEMA; (c) PAEAHPMA; (d) PAEAEMA.
Fig. 9. Luciferase expression in 293T cells mediated by different polyplexes: (a) PAHPMA, (b) PAEMA, (c) PAEAHPMA, (d) PAEAEMA at N/P = 20, 30 and 40 and (e) PEI at N/P = 10 in serum-free DMEM media.
Fig. 7. Heparin displacement assay with (a) PAHPMA/DNA complexes, (b) PAEMA/ DNA complexes, (c) PAEAHPMA/DNA complexes and (d) PAEAEMA/DNA complexes at N/P = 20 incubated with increasing concentrations of heparin for 30 min at room temperature.
gene vector, was used for comparison. As shown in Fig. 9, PAHPMA showed a lower transfection efficiency than PAEMA at all N/P ratios tested. In comparison with PAEMA, with a similar chemical structure, the lower gene transfection efficiency of PAHPMA might be due to the introduction of hydroxyl groups, i.e. the existence of hydroxyl groups on PAHPMA could increase its binding capacity for DNA and at the same time decrease the surface charge of the polymer/DNA complexes due to the formation of hydrogen bonds
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Acknowledgements This work was financially supported by the National Natural Science Foundation of China (20704032), National Basic Research Program of China (2009CB930300).
References
Fig. 10. Luciferase expression in 293T cells mediated by different polyplexes: (a) PAHPMA, (b) PAEMA, (c) PAEAHPMA, (d) PAEAEMA at N/P = 20, 30 and 40 and (e) PEI at N/P = 10 in 10% serum-supplemented DMEM media.
between the polymers and DNA. Thus, a lower zeta potential and stronger binding ability may result in the lower gene transfection efficiency of PAHPMA than PAEMA. Comparing the results for PAEAHPMA and PAEAEMA, we found that the PAEAHPMA/DNA complexes possessed a similar transfection efficiency to that of PAEAEMA/DNA complexes, which might mean that the effect of hydroxyl groups on gene transfection efficiency was weakened when ethylenediamine instead of ammonia was introduced into the sidechains of PGMA. Nevertheless, further studies are necessary to elucidate the exact role of the hydroxyl groups in polyplex formation and the correlation between binding affinity of the polymers for pDNA and their gene transfection efficiency. In vitro transfection efficiency was further studied in DMEM with 10% serum. As showed in Fig. 10, PAEMA and PAEAEMA showed a slightly lower transfection efficiency than in the absence of serum, while PAHPMA and PAEAHPMA retained similar transfection efficiencies. This implies that the shielding effect of hydroxyl groups might maintain a high transfection efficiency of the corresponding polymer/DNA complexes in the presence of serum. 4. Conclusions In this study four cationic polymethacrylates, PAEMA, PAHPMA, PAEAEMA and PAEAHPMA, with similar chemical structures and degrees of polymerization, were synthesized by ATRP to evaluate the effect of hydroxyl groups on the relative properties of cationic polymeric gene vectors. The results of a heparin displacement assay showed that hydroxyl groups on the sidechains of PAHPMA might facilitate DNA binding, but data from a gel retardation assay indicated that there were no obvious differences when hydroxylated polymers were used. The other results showed that PAHPMA and PAEAHPMA possessed lower zeta potentials and lower cytotoxicities than PAEMA and PAEAEMA, respectively, due to the introduction of hydroxyl groups into PAHPMA and PAEAHPMA. In vitro gene transfection studies in 293T cells without serum showed that PAHPMA exhibited a lower transfection efficiency than PAEMA, and PAEAHPMA/DNA complexes possessed a similar transfection efficiency to PAEAEMA/DNA complexes, whereas PAHPMA and PAEAHPMA retained similar transfection efficiencies in DMEM with 10% serum. The reason for this might be that the existence of hydroxyl groups increases the binding capacity with DNA and at the same time decreases the surface charge of the polymer/ DNA complexes due to the formation of hydrogen bonds between the polymer and DNA. Therefore, a lower zeta potential and stronger binding ability maybe result in a lower gene transfection efficiency. This effect of hydroxyl groups decreases with increasing amino group density on the polymer.
[1] Morille M, Passirani C, Vonarbourg A, Clavreul A, Benoit JP. Progress in developing cationic vectors for non-viral systemic gene therapy against cancer. Biomaterials 2008;29:3477–96. [2] Nguyen DN, Green JJ, Chan JM, Langer R, Anderson DG. Polymeric materials for gene delivery and DNA vaccination. Adv Mater 2009;21:847–67. [3] Mintzer MA, Simanek EE. Nonviral vectors for gene delivery. Chem Rev 2009;109:259–302. [4] Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 1995;92:7297–301. [5] Zou SM, Erbacher P, Remy JS, Behr JP. Systemic linear polyethylenimine (LPEI)-mediated gene delivery in the mouse. J Gene Med 2000;2:128–34. [6] Sonawane ND, Szoka FC, Verkman AS. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine–DNA polyplexes. J Biol Chem 2003;278:44826–31. [7] Dubruel P, Schacht E. Vinyl polymers as non-viral gene delivery carriers: current status and prospects. Macromol Biosci 2006;6:789–810. [8] Wagner E, Cotton M, Foisner R, Birnstiel ML. Transferrin–polycation–DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proc Natl Acad Sci USA 1991;88:4255–9. [9] Haensler J, Szoka Jr FC. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjug Chem 1993;4:372–9. [10] Schaffer DV, Fidelman NA, Dan N, Lauffenburger DA. Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery. Biotechnol Bioeng 2000;67:598–606. [11] Goncalves C, Mennesson E, Fuchs R, Gorvel JP, Midoux P, Pichon C. Macropinocytosis of polyplexes and recycling of plasmid via the clathrindependent pathway impair the transfection efficiency of human hepatocarcinoma cells. Mol Ther 2004;10:373–85. [12] Curiel DT, Agarwal S, Wagner E, Cotton M. Adenovirus enhancement of transferrin–polylysine-mediated gene delivery. Proc Natl Acad Sci USA 1991;88:8850–4. [13] Lukacs GL, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman AS. Sizedependent DNA mobility in cytoplasm and nucleus. J Biol Chem 2002;275:1625–9. [14] Ludtke JJ, Zhang GF, Sebestyen MG, Wolff JA. A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA. J Cell Sci 1999;112:2033–41. [15] Brunner S, Furtbauer E, Sauer T, Kursa M, Wagner E. Overcoming the nuclear barrier: cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol Ther 2002;5:80–6. [16] Merdan T, Kunath K, Petersen H, Bakowsky U, Voigt KH, Kopecek J, et al. PEGylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice. Bioconjug Chem 2005;16:785–92. [17] Li J, Yang C, Li H, Wang X, Goh SH, Ding JL, et al. Cationic supramolecules composed of multiple oligoethylenimine-grafted beta-cyclodextrins threaded on a polymer chain for efficient gene delivery. Adv Mater 2006;18:2969–74. [18] Yang C, Wang X, Li H, Tan E, Lim CT, Li J. Cationic polyrotaxanes as gene carriers: physicochemical properties and real-time observation of DNA complexation, and gene transfection in cancer cells. J Phys Chem B 2009;113:7903–11. [19] Wang DA, Narang AS, Kotb M, Gaber AO, Miller DD, Kim SW, et al. Novel branched poly(ethylenimine)–cholesterol water-soluble lipopolymers for gene delivery. Biomacromolecules 2002;3:1197–207. [20] Choi JS, Nam K, Park JY, Kim JB, Lee JK, Park JS. Enhanced transfection efficiency of PAMAM dendrimer by surface modification with L-arginine. J Control Release 2004;99:445–56. [21] Gao X, Kuruba R, Damodaran K, Day BW, Liu D, Li S. Polyhydroxylalkyleneamines: a class of hydrophilic cationic polymer-based gene transfer agents. J Control Release 2009;137:38–45. [22] Liu YM, Reineke TM. Hydroxyl stereochemistry and amine number within poly(glycoamidoamine)s affect intracellular DNA delivery. J Am Chem Soc 2005;127:3004–15. [23] Liu YM, Reineke TM. Poly(glycoamidoamine)s for gene delivery: stability of polyplexes and efficacy with cardiomyoblast cells. Bioconjug Chem 2006;17:101–8. [24] Liu YM, Wenning L, Lynch M, Reineke TM. New poly(D-glucaramidoamine)s induce DNA nanoparticle formation and efficient gene delivery into mammalian cells. J Am Chem Soc 2004;126:7422–3. [25] Xu FJ, Li HZ, Li J, Zhang ZX, Kang ET, Neoh KG. Pentablock copolymers of poly(ethylene glycol), poly((2-dimethyl amino)ethyl methacrylate) and poly(2-hydroxyethyl methacrylate) from consecutive atom transfer radical polymerizations for non-viral gene delivery. Biomaterials 2008;29:3023–33.
M. Ma et al. / Acta Biomaterialia 6 (2010) 2658–2665 [26] Tziveleka LA, Psarra AMG, Tsiourvas D, Paleos CM. Synthesis and characterization of guanidinylated poly(propylene imine) dendrimers as gene transfection agents. J Control Release 2007;117:137–46. [27] Geall AJ, Taylor RJ, Earll ME, Eaton MAW, Blagbrough IS. Synthesis of cholesteryl polyamine carbamates: pK(a) studies and condensation of calf thymus DNA. Bioconjug Chem 2000;11:314–26. [28] Dubruel P, Christiaens B, Rosseneu M, Vandekerckhove J, Grooten J, Goossens V, et al. Buffering properties of cationic polymethacrylates are not the only key to successful gene delivery. Biomacromolecules 2004;5:379–88.
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Influence of hydroxyl groups on the biological properties of cationic polymethacrylates as gene vectors Ming Ma, Feng Li *, Zhe-fan Yuan, Ren-xi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education, College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 4 August 2009 Received in revised form 10 January 2010 Accepted 15 January 2010 Available online 22 January 2010 Keywords: Cationic polymers Gene delivery Hydroxyl Atom transfer radical polymerization
a b s t r a c t In this study poly(aminoethyl methacrylate) (PAEMA), poly(3-amino-2-hydroxypropyl methacrylate) (PAHPMA), poly(2-(2-aminoethylamino)ethyl methacrylate) (PAEAEMA) and poly(3-(2-aminoethylamino) 2-hydroxypropyl methacrylate) (PAEAHPMA) were synthesized using atom transfer radical polymerization to evaluate the effect of hydroxyl groups on the relative properties of cationic polymeric gene vectors. The results of heparin displacement assays showed that PAHPMA possessed a stronger binding capacity than PAEMA. PAHPMA/DNA complexes and PAEAHPMA/DNA complexes had lower zeta potentials than those of PAEMA and PAEAEMA. MTT assay results indicated that PAHPMA and PAEAHPMA exhibited obviously lower cytotoxicities than PAEMA and PAEAEMA. Subsequently, in vitro gene transfection studies in 293T cells without serum showed that PAHPMA exhibited a lower transfection efficiency than PAEMA and PAEAHPMA/DNA complexes possessed a similar transfection efficiency to PAEAEMA/ DNA complexes. Moreover, PAHPMA and PAEAHPMA retained similar transfection efficiencies in DMEM with 10% serum, but PAEMA and PAEAEMA showed slightly lower transfection efficiencies than in the absence of serum. The reason for these phenomena might be attributed to the introduction of hydroxyl groups into PAHPMA and PAEAHPMA, i.e. the existence of hydroxyl groups might increase the binding capacity to DNA and at the same time decrease the surface charge of the polymer/DNA complexes due to the formation of hydrogen bonds between the polymers and DNA. Therefore, a lower zeta potential and stronger binding ability may result in a lower gene transfection efficiency. This effect of hydroxyl groups decreased with increasing amino group density on the polymer. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Non-viral vectors have attracted more and more attention due to their low immune response and low antigenicity, as well as simplicity of preparation [1–3]. Cationic polymers, such as polyethyleneimines (PEIs), poly(dimethylamino ethyl methacrylate) (PDMAEMA), poly(L-lysine) (PLL), and polyamidoamines [4–9], are the major type investigated in the past decades. However, most polycation vectors suffer from unsatisfactory transfection efficiencies and have remained at the laboratory stage until now. Ideal non-viral vectors should be non-toxic, form compact DNA particles of virus-like dimensions, protect the DNA from degradation and facilitate targeted cell binding and internalization, endosomal escape, trafficking the cytoplasmic environment and localizing into the nucleus, as well as vector unpacking [10–15]. In reality, no polycation is able to carry out all the extracellular and intracellular delivery functions. To improve the transfection efficiency of polycation vectors additional functional elements, e.g. poly(ethylene glycol), cyclodextrins, cholesterol, L-arginine and/or hydroxyl groups, need * Corresponding author. Tel./fax: +86 27 68754509. E-mail addresses: [email protected], [email protected] (F. Li).
to be incorporated to modify the polymers such that they exhibit different biological characteristics from unmodified ones. This indicates that subtle differences in chemical structure can have a significant impact on the process of transfection [16–21]. For example, Reineke detailed the effect of hydroxyl groups on the process of gene delivery based on a new library of poly(glycoamidoamine)s synthesized by condensation polymerization of esterified D-glucaric acid, dimethyl meso-galactarate, D-mannaro-1,4:6,3-dilactone and dimethyl L-tartarate with oligoamines. The results showed that the poly(glycoamidoamine) structures containing four secondary amines between the carbohydrate repeat units delivered plasmid DNA containing the firefly luciferase reporter gene with high efficacy into a variety of mammalian cell lines (BHK-21, HeLa and HepG2 cells) in a non-toxic manner, and the biological properties, particularly the delivery efficiency, of these polymers were affected by the hydroxyl groups and their stereo structure together [22–24]. Until now many groups have reported that the inclusion of hydroxyl groups in polycations has helped reduce cytotoxicity and improved gene delivery efficiency. For example, Kang found that incorporating poly(2-hydroxyethyl methacrylate) into poly(dimethylaminoethyl methacrylate) block polymers could enhance transfection efficiency due to the good biocompatibility and shielding effect of the
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.01.024
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poly(2-hydroxyethyl methacrylate) blocks [25]. However, Tziveleka indicated that a fourth generation polypropyleneimine dendrimer lost its transfetion efficiency when the primary amino groups in the dendrimer were completely replaced with hydroxylated moieties [26]. Thus, a better understanding of such structure–bioactivity relationships is required. Here we synthesized four polymers, poly(aminoethyl methacrylate) (PAEMA), poly(3-amino-2-hydroxypropyl methacrylate) (PAHPMA), poly(2-(2-aminoethylamino) ethyl methacrylate) (PAEAEMA) and poly(3-(2-aminoethylamino) 2-hydroxypropyl methacrylate) (PAEAHPMA), with similar chemical structures to evaluate their differences in a number of parameters, such as binding capacity and gene delivery. This study will be helpful in further elucidating the effect of hydroxyl groups on the biological properties of polymeric vectors.
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2.4. Synthesis of PAEAHPMA PGMA (0.1 g) and Boc-EDA (2.3 g) were dissolved in 50 ml of DMF and reacted at 70 °C for 4 h. After the reaction the mixture was added to 5 M HCl and stirred for 5 h, and then dialyzed against excess HCl solution (2 l, pH 3) with three changes of acid a day for 2 days. The polymer was obtained by freeze drying. 2.5. Synthesis of PAEMA and PAEAEMA The Boc-protected PAEMA and Boc-PAEAEMA were prepared by a similar approach to the synthesis of PGMA at the same initiator/ monomer ratio (1:70). The resultant polymer was stirred in 5 M HCl for 3–5 h. After the signal for tert-butoxycarbonyl groups completely disappeared from 1H NMR spectra the solution was dialyzed for 2 days. PAEMA and PAEAEMA were collected after freeze drying.
2. Materials and methods 2.1. Materials
2.6. Polymer characterizations 1
Glycidyl methacrylate (GMA) (Aldrich, 97% pure) was purified by vacuum distillation under reduced pressure and stored in a refrigerator under a nitrogen atmosphere. N-(2-hydroxyethyl)ethlyenediamine was purchased from Alfa. Copper (I) bromide (Beijing Chemical Co.) was purified by washing with acetic acid and ethanol in turn. Ammonia (27%), methacryloyl chloride, ethanolamine, ethylenediamine, 2,2-bipyridine (Bpy), N,N-dimethylformamide (DMF), dioxane, dichloromethane and ether were all purchased from Beijing Chemical Co. and used without further purification. N-tert-butoxycarbonyl-ethylenediamine (Boc-EDA) and N-tert-butoxycarbonyl-aminoethyl methacrylate (Boc-AEMA) were prepared according to Geall et al. [27] and Dubruel et al. [28]. N,N0 -di-(tert-butoxycarbonyl)-2-(2-aminoethylamino)ethyl methacrylate (Boc-AEAEMA) was synthesized via a similar method to Boc-AEMA. Ethyl 2-bromoisobutyrate (EBIB) (98% pure), Dubelcco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, trypsin, phosphate-buffered saline (PBS) and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich. The reporter plasmid pGL3-Luc was purchased from Promega. The plasmid DNA was stored at 20 °C prior to use.
H nuclear magnetic resonance (NMR) spectrometry (Mercury VX-300) and Fourier transform infrared (FT-IR) spectrometry (AVATAR 360) were used to determine the chemical structures of the synthesized polymers in either CDCl3 or deuterated water, while the molecular weights and molecular weight distributions were estimated by gel permeation chromatography (GPC) using a Waters 2690 separation module and a Waters 2410 refractive index detector, with HAc–NaAc buffer solution (0.03 mol l1, pH 3) as eluent and poly(ethylene glycol) with a narrow molecular distribution as the standard. Transmission electron microscopy (TEM) experiments were performed in a JEM-2010 instrument operating at an acceleration voltage of 200 keV. 2.7. Cell culture 293T cells were incubated in DMEM supplemented with 10 vol.% fetal bovine serum (FBS) and antibiotics (10,000 U ml1 penicillin–streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were subcultured prior to confluence using trypsin–EDTA. 2.8. DNA binding
2.2. Synthesis of poly(glycidyl methacrylate) (PGMA) GMA (3.5 g, 24 mmol), EBIB (0.068 g, 0.352 mmol) and Bpy (0.11 g, 0.7 mmol) were dissolved in 4 ml of DMF in a brown reaction flask and degassed by three freeze–pump–thaw cycles, then CuBr (0.05 g, 0.352 mmol) was added. After another three freeze– pump–thaw cycles polymerization was started under an Ar atmosphere at 60 °C for 7 h. After that the solution was poured into excess ether to precipitate the polymer. The precipitate was dissolved in dichloromethane and purified using an alumina column to eliminate the used copper, then precipitated again in ether. The polymer obtained was dried in vacuum.
2.3. Synthesis of PAHPMA PGMA (0.1 g) was dissolved in dioxane and ammonia (2:1 vol.%) mixed solution in a sealed reactor and reacted at 30 °C for 7 days. Subsequently the mixture was concentrated and dialyzed against excess HCl solution (2 l, pH 3) with three changes of acid a day for 2 days. The polymer was obtained by freeze drying.
The DNA condensation ability of the polymers was assessed by means of an agarose gel retardation assay. Polycation/pDNA complexes with different N/P ratios from 0 to 7 were formed by adding an appropriate volume of polymer solution (150 mM NaCl) to 0.1 lg of pDNA (0.1 mg ml1 in 40 mM Tris–HCl buffer). Then 6 ll of sample containing 1 ll of ethidium bromide and 0.1 lg of DNA were electrophoresed through a 1% agarose gel using Tris–borate–EDTA buffer (pH 7.2) at 80 mV for 80 min. DNA retardation was observed by irradiation with UV light and assayed using Cam2com software. 2.9. Particle size and zeta-potential measurements The particle size and zeta potential were determiner in a NanoZSZEN3600 (Malvern) instrument at room temperature. The polycation/pDNA complexes were prepared according to the previous procedure at N/P ratios ranging from 10 to 50. Then the polyplexes were incubated at room temperature for 30 min. After that the polyplexes were diluted with 150 mM NaCl solution or pure water to 1.0 ml volume for size and zeta-potential measurements, respectively.
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2.10. Heparin displacement The binding intensity of polyplexes was determined by heparin displacement experiments. The sample/DNA complexes were first prepared at an N/P ratio of 20 for 30 min as described above and then incubated with heparin solutions of different concentration (ranging from 0.1 to 2 mg ml1). An agarose gel retardation assay was used to detect the released DNA. 2.11. Cytotoxicity assay The cytotoxicity assay was carried out on the basis of an MTT assay in 293T cells. Cells were seeded in 96-well plates at an initial density of 6000 cells well1 and cultured for 24 h in 200 ll of DMEM. 24 h after polymer was added the medium was replaced with MTT reagent (20 ll in PBS at 5 mg ml1) and then incubated for another 4 h. Thereafter MTT was removed and 200 ll of DMSO was added until the crystals dissolved. The absorbance at 570 nm of the solution in each well was recorded using a Microplate Reader (Bio-Rad model 550). Cell viability was calculated according to the equation: cell viability (%) = (ODsample ODblank)/(ODcontrol ODblank) 100, where ODsample is the absorbance of the solution of cells cultured with the polymer or PEI, ODblank is the absorbance of the medium and ODcontrol is the absorbance of the solution of cells cultured with medium only. 2.12. In vitro transfection For transfection experiments pGL3 plasmid DNA was used to evaluate the transfection efficiency of the four polymers in 293T cells. 293T cells were seeded in 24-well plates at a density of 6 104 cells well1 and cultured with 1 ml of DMEM containing 10% FBS for 1 day at 37 °C in a humidified atmosphere of 95% air and 5% CO2 until the cells reached 70% confluence. The polymer/DNA complexes were formed at different N/P ratios ranging from 20 to 40 according to the conditions described above (containing 1 lg DNA for each unit N/P ratio). The complexes were added to each well and incubated in 1 ml of DMEM at 37 °C for 4 h, then the medium was replaced with 1 ml of DMEM containing 10% FBS and the cells were further incubated for 48 h. After incubation the cells were permeabilized with 200 ll well1 cell lysis buffer. Luciferase activity was measured by detecting the light emission from an aliquot of cell lysate incubated with 100 ll of luciferin substrate (Promega) in a luminometer (Lumat LB9507, Berthold). The light units measured using protein assay kits (Pierce) were normalized against the protein concentration in the cell extracts. Luciferase activity was expressed as relative light units (RLU mg protein1). The procedures for transfection experiments in serum were as described above except that the medium was replaced by DMEM supplemented with 10% FBS.
The 1H NMR spectrum of PGMA is shown in Fig. 1. The peaks at 0.93–1.09 and 1.92–1.97 p.p.m. were ascribed to the methyl and methylene protons on the backbone of PGMA. The peaks at 2.64, 2.84 and 3.23 p.p.m. are characteristic of the epoxy groups on the sidechain of PGMA, and the peaks at 3.79 and 4.42 p.p.m. were assigned to the methylene protons adjacent to the epoxy group for space isomerization of the epoxy group. After nucleophilic reaction of the amines with epoxy groups of PGMA the signal for the epoxy groups completely disappeared and the peak of the methylene protons adjacent to the epoxy groups shifted to 3.92–4.20 p.p.m. for PAHPMA and 3.72–3.99 p.p.m. for PAEAHPMA. The signal for methine connected to hydroxyls also occurred here. The peaks at 2.91–3.50 p.p.m. for PAHPMA and 2.68–3.29 p.p.m. for PAEAHPMA belonged to the characteristic peaks of methylene protons adjacent to amino groups. The ring-opening reaction of epoxy groups was further confirmed by FT-IR (Fig. 2). The characteristic absorptions of the epoxide ring at 849 and 907 cm1 also disappeared after the ring-opening reaction and a broad band at 3431 cm–1, assigned to N–H stretching and O–H stretching, appeared. PAEMA and PAEAEMA possessed similar chemical shift peaks to PAHPMA and PAEAHPMA, except that of methylene protons adjacent to hydroxyl groups. Moreover, disappearance of the peak at 1.41 p.p.m. in the 1 H NMR spectra indicated the entire elimination of protected groups (data not shown). The molecular weights and molecular weight distributions of all polymers were also evaluated by GPC using HAc–NaAc buffer solution as the eluent. According to the results of GPC, the number average molecular weights of PAHPMA, PAEAHPMA, PAEMA and PAEAEMA were 8600, 17,000, 10,000 and 14,500 g mol1, respectively. Thus, the repeating units of these four polymers were 62, 85, 76 and 80, respectively, according to the GPC results. And their molecular weight distributions were 1.99, 2.4, 1.95 and 1.33. The above results indicate that the desired polymers were successfully prepared. 3.2. Gel retardation assay The ability of polymers to condense DNA was first investigated by gel retardation assay. The amount of polymer added was calculated on the basis of the chosen N/P ratio, i.e. polymer nitrogen atoms to DNA phosphorus atoms. As shown in Fig. 3, all polymers exhibited good DNA binding ability. PAHPMA and PAEMA retarded bulk DNA movement at a similar N/P ratio. It seems that there was no obvious difference in the N/P ratio needed when hydroxylated polymers were used. Furthermore, PAEAHPMA and PAEAEMA showed a similar result. However, according to the earlier literature increasing numbers of hydroxyl groups could hinder interaction with DNA due to the neutral functionality [23,31]. Thus, additional experiments to further evaluate the effect of hydroxyl groups on DNA binding will be carried out by heparin displacement assay below. 3.3. Particle size and zeta-potential measurements
3. Results and discussion 3.1. Synthesis of polymers Atom transfer radical polymerization (ATRP), one of the most actively developing areas in polymer chemistry, is a powerful tool for the preparation of well-defined polymers with predetermined molecular weights and molecular weight distributions [29,30]. Thus, in order to synthesize PAHPMA, PAEAHPMA, PAEMA and PAEAEMA with similar degrees of polymerization, ATRP was used to polymerize GMA, Boc-AEMA and Boc-AEAEMA at the same initiator/monomer ratio (1:70). 1H NMR, FT-IR and GPC were used to characterize these polymers (Scheme 1).
The particle sizes of polyplexes were investigated in 150 mM NaCl solution by dynamic light scattering (DLS). As revealed in Fig. 4, all polycations could condense DNA into nanoparticles to be endocytosed (ranging from 157 ± 10 to 381 ± 10 nm). The PAHPMA/DNA and PAEMA/DNA complexes showed a trend of decreasing average diameter with increasing N/P ratio from 10 to 50. No distinct difference in size was observed at parallel N/P ratios for the two polycations/DNA complexes. A similar trend was also observed for the PAEAHPMA/DNA and PAEAEMA/DNA complexes, except that the average size of the PAEAHPMA/DNA complexes was smaller than that of the PAEAEMA/DNA complexes at the same N/P ratio in the range 10–30. It seems that hydroxyl groups on
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Scheme 1. Schematic illustration of PAHPMA, PAEMA, PAEAHPMA and PAEAEMA.
Fig. 1. 1H NMR spectra of PGMA (in CDCl3), PAHPMA and PAEAHPMA (in D2O).
Fig. 2. FT-IR spectra of (a) PGMA, (b) PAHPMA and (c) PAEAHPMA.
PAHPMA and PAEAHPMA had no obvious effect on the size of the polyplexes.
TEM was then used to characterize the morphology of the PAHPMA/DNA and PAEMA/DNA complexes at a N/P ratio of 20. As shown in Fig. 5, both polycations condensed DNA into nanoparticles with a diameter of 300 ± 30 nm. There was no significant difference in size, as in the DLS results. However, the particle sizes measured by TEM were appreciably smaller than those obtained by DLS. This might be because the particle sizes measured by DLS were obtained in the hydrated state in solution, while those obtained by TEM had been dried after dropping polyplexe solution onto carbon-coated copper meshes. The zeta potentials of the polyplexes were measured in pure water, and are shown in Fig. 6. It is obvious that the PAHPMA/ DNA and PAEAHPMA/DNA complexes showed much lower zeta potentials than those of PAEMA and PAEAEMA, respectively. This effect is not as yet clearly understood; we presume that this may be ascribed to the stronger intermolecular hydrogen bonding between the polymers with hydroxyl groups and DNA, which resulted in lower zeta potential values [22]. This is not the same as the case of poly(ethylene glycol) (PEG), because PEG mainly reduces the zeta potential of polyplexes by steric hindrance [16]. The zeta potential of PAHPMA/DNA complexes increased from 3.6 ± 0.2 to 8.6 ± 1.2 mV on increasing the N/P ratio from 10 to
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Fig. 4. Particle sizes of (a) PAHPMA/DNA complexes, (b) PAEMA/pDNA complexes, (c) PAEAHPMA/DNA complexes and (d) PAEAEMA/DNA complexes measured by DLS in 150 mM NaCl solution.
were obviously disrupted at a heparin concentration of 1 mg ml1. This indicates that the hydroxyl groups on the sidechains of PAHPMA might facilitate DNA binding. Furthermore, the zeta potential of PAHPMA/DNA complexes at N/P = 20 was much lower than that of PAEMA/DNA complexes (4.6 ± 0.2 vs. 13.4 ± 1.2 mV), which suggests that the lower surface charge of the PAHPMA/DNA complexes might be less attractive to negatively charged heparin, resulting in less replacement of DNA. This may be another explanation for the above heparin displacement results. The PAEAHPMA/DNA complexes exhibited obvious disassembly at 1 mg ml–1 heparin, like PAEAEMA, but disassociation was weaker than that of PAEAEMA/ DNA complexes at the same heparin concentration. This might indicate that PAEAHPMA possesses a slightly stronger binding ability than PAEAEMA on the one hand, while, on the other hand, the shielding effect of hydroxyl groups decreases, in comparison with PAHPMA, with the introduction of secondary amino groups into PAEAHPMA. The above results indicate that the presence of hydroxyl groups in PAHPMA and PAEAHPMA facilitate DNA binding due to the possible formation of hydrogen bonds between hydroxyl groups on the polymers and DNA base pairs [32]. However, the detailed mechanism of binding and the shielding effect of hydroxyl groups is not yet understood and requires further study. Fig. 3. DNA binding ability of polymers. Electrophoretic mobility of plasmid DNA in (a) PAHPMA, (b) PAEMA, (c) PAEAHPMA and (d) PAEAEMA/DNA complexes at the N/P ratios specified (0–7).
50, while PAEAHPMA/DNA maintained an almost steady zeta potential around 30 mV. The sharp difference in zeta potentials between them might be due to the higher amino group density for PAEAHPMA, which contains additional secondary amino groups. Thus, these results might show that the existence of hydroxyl groups can shield the surface charge of complexes due to the formation of hydrogen bonds and that the shielding effect of hydroxyl groups decreases with increasing amino group density of the polymer. 3.4. Heparin displacement assay To further investigate the binding effect of hydroxyl groups, a heparin displacement assay was carried out, with the N/P ratio for all the polycation/DNA complexes being kept at 20. As revealed in Fig. 7, PAHPMA/DNA complexes remained almost stable up to a heparin concentration of 2 mg ml1, while PAEMA/DNA complexes
3.5. Cytotoxicity of the polymers The cytotoxicity of the four polymers was evaluated in 293T cells by MTT assay. As shown in Fig. 8, PAHPMA and PAEAHPMA exhibited obviously higher relative cell viability than PAEMA and PAEAEMA, and PAHPMA showed 55.45% relative cell viability even when the polymer concentration was 2.75 mg ml1. One probable reason for the lower cytotoxicity of PAHPMA and PAEAHPMA might be their hydroxyl groups, which enhance the biocompatibility of polycations [22]. 3.6. In vitro transfection Gene transfection efficiency was influenced by many factors, such as binding capacity and biocompatibility of the polymers, surface charge and the size of the corresponding polymer/DNA complexes. The above results show that hydroxyl groups on the sidechains of PAHPMA and PAEAHPMA have a significant impact on their properties. These changes may induce different gene transfection efficiencies. Here, 293T cells were used to evaluate the effect of hydroxyl groups on gene transfection activity. Branched 25 kDa PEI, which is the most widely used polycation
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Fig. 5. TEM images of (a) PAHPMA/DNA complexes (b) PAEMA/DNA complexes at N/P = 20.
Fig. 6. Zeta potentials of (a) PAHPMA/DNA complexes, (b) PAEMA/pDNA complexes, (c) PAEAHPMA/DNA complexes and (d) PAEAEMA/DNA complexes at various N/P ratios.
Fig. 8. Relative cell viability of 293T cells co-incubated with polymers for 24 h: (a) PAHPMA; (b) PAEMA; (c) PAEAHPMA; (d) PAEAEMA.
Fig. 9. Luciferase expression in 293T cells mediated by different polyplexes: (a) PAHPMA, (b) PAEMA, (c) PAEAHPMA, (d) PAEAEMA at N/P = 20, 30 and 40 and (e) PEI at N/P = 10 in serum-free DMEM media.
Fig. 7. Heparin displacement assay with (a) PAHPMA/DNA complexes, (b) PAEMA/ DNA complexes, (c) PAEAHPMA/DNA complexes and (d) PAEAEMA/DNA complexes at N/P = 20 incubated with increasing concentrations of heparin for 30 min at room temperature.
gene vector, was used for comparison. As shown in Fig. 9, PAHPMA showed a lower transfection efficiency than PAEMA at all N/P ratios tested. In comparison with PAEMA, with a similar chemical structure, the lower gene transfection efficiency of PAHPMA might be due to the introduction of hydroxyl groups, i.e. the existence of hydroxyl groups on PAHPMA could increase its binding capacity for DNA and at the same time decrease the surface charge of the polymer/DNA complexes due to the formation of hydrogen bonds
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Acknowledgements This work was financially supported by the National Natural Science Foundation of China (20704032), National Basic Research Program of China (2009CB930300).
References
Fig. 10. Luciferase expression in 293T cells mediated by different polyplexes: (a) PAHPMA, (b) PAEMA, (c) PAEAHPMA, (d) PAEAEMA at N/P = 20, 30 and 40 and (e) PEI at N/P = 10 in 10% serum-supplemented DMEM media.
between the polymers and DNA. Thus, a lower zeta potential and stronger binding ability may result in the lower gene transfection efficiency of PAHPMA than PAEMA. Comparing the results for PAEAHPMA and PAEAEMA, we found that the PAEAHPMA/DNA complexes possessed a similar transfection efficiency to that of PAEAEMA/DNA complexes, which might mean that the effect of hydroxyl groups on gene transfection efficiency was weakened when ethylenediamine instead of ammonia was introduced into the sidechains of PGMA. Nevertheless, further studies are necessary to elucidate the exact role of the hydroxyl groups in polyplex formation and the correlation between binding affinity of the polymers for pDNA and their gene transfection efficiency. In vitro transfection efficiency was further studied in DMEM with 10% serum. As showed in Fig. 10, PAEMA and PAEAEMA showed a slightly lower transfection efficiency than in the absence of serum, while PAHPMA and PAEAHPMA retained similar transfection efficiencies. This implies that the shielding effect of hydroxyl groups might maintain a high transfection efficiency of the corresponding polymer/DNA complexes in the presence of serum. 4. Conclusions In this study four cationic polymethacrylates, PAEMA, PAHPMA, PAEAEMA and PAEAHPMA, with similar chemical structures and degrees of polymerization, were synthesized by ATRP to evaluate the effect of hydroxyl groups on the relative properties of cationic polymeric gene vectors. The results of a heparin displacement assay showed that hydroxyl groups on the sidechains of PAHPMA might facilitate DNA binding, but data from a gel retardation assay indicated that there were no obvious differences when hydroxylated polymers were used. The other results showed that PAHPMA and PAEAHPMA possessed lower zeta potentials and lower cytotoxicities than PAEMA and PAEAEMA, respectively, due to the introduction of hydroxyl groups into PAHPMA and PAEAHPMA. In vitro gene transfection studies in 293T cells without serum showed that PAHPMA exhibited a lower transfection efficiency than PAEMA, and PAEAHPMA/DNA complexes possessed a similar transfection efficiency to PAEAEMA/DNA complexes, whereas PAHPMA and PAEAHPMA retained similar transfection efficiencies in DMEM with 10% serum. The reason for this might be that the existence of hydroxyl groups increases the binding capacity with DNA and at the same time decreases the surface charge of the polymer/ DNA complexes due to the formation of hydrogen bonds between the polymer and DNA. Therefore, a lower zeta potential and stronger binding ability maybe result in a lower gene transfection efficiency. This effect of hydroxyl groups decreases with increasing amino group density on the polymer.
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