PGMA-Based Cationic Nanoparticles with Polyhydric Iodine Units for

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PGMA-Based Cationic Nanoparticles with Polyhydric Iodine Units for Advanced Gene Vectors Yue Sun,†,‡,§ Hao Hu,†,‡,§ Bingran Yu,*,†,‡,§ and Fu-Jian Xu*,†,‡,§ †

State Key Laboratory of Chemical Resource Engineering, ‡Key Laboratory of Carbon Fiber and Functional Polymers, Ministry of Education, and §Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 China S Supporting Information *

ABSTRACT: It is crucial for successful gene delivery to develop safe, effective, and multifunctional polycations. Iodine-based small molecules are widely used as contrast agents for CT imaging. Herein, a series of star-like poly(glycidyl methacrylate) (PGMA)based cationic vectors (II-PGEA/II) with abundant flanking polyhydric iodine units are prepared for multifunctional gene delivery systems. The proposed II-PGEA/II star vector is composed of one iohexol intermediate (II) core and five ethanolamine (EA) and II-difunctionalized PGMA arms. The amphipathic II-PGEA/II vectors readily self-assemble into welldefined cationic nanoparticles, where massive hydroxyl groups can establish a hydration shell to stabilize the nanoparticles. The II introduction improves cell viabilities of polycations. Moreover, by controlling the suitable amount of introduced II units, the resultant II-PGEA/II nanoparticles can produce fairly good transfection performances in different cell lines. Particularly, the II-PGEA/II nanoparticles induce much better in vitro CT imaging abilities in tumor cells than iohexol (one commonly used commercial CT contrast agent). The present design of amphipathic PGMA-based nanoparticles with CT contrast agents would provide useful information for the development of new multifunctional gene delivery systems.

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INTRODUCTION Nonviral vectors are promising for effective gene delivery due to their good immunocompatibility and transfection performance.1−5 Compared with liposome, polycations draw much more attention because of their simple modification and flexible design strategies.6−10 A number of polycations, such as polyethylenimine (PEI),11,12 polyamidoamine,13 poly(2-(dimethylamino) ethyl methacrylate),14,15 and poly(L-lysine),16 exhibit some favorable properties in gene delivery applications. However, the high cytotoxicity caused by positive charges is still an obstacle to the application of polycations. Ring-opening reaction is a promising method to produce biomedical materials with various functions and good biocompatibility.17−19 Epoxy groups could be considered as potential reagents for biocompatible materials. Hydrophilic ether bonds or hydroxyl groups can be formed by ring opening of epoxy units. The resulting hydration shell (a layer between the surface of the designed structures and the surrounding water) could improve the biocompatibility or shield the static charges of the modified materials or drug/gene delivery systems.20−22 Atom transfer radical polymerization (ATRP) is a well-controlled living polymerization to prepare polymers with designed molecular weights and flexile molecular structures.23−25 Glycidyl methacrylate (GMA) is a good monomer for ATRP, and the resultant PGMA is readily postmodified by ring-opening reactions. It was reported that © XXXX American Chemical Society

ethanolamine (EA)-modified PGMA (denoted by PGEA) provides good chances to develop versatile types of gene vectors with plentiful hydroxyl groups.26−28 It is very important for promising gene delivery systems to develop multifunctional polycations. With the development of clinical medicine, computed tomography (CT) already comes to a universal method in disease diagnosis fields.29,30 Iodinebased small molecules such as iohexol, iopromide, and iodixanol are widely used as contrast agents for CT imaging.31,32 However, small molecule agents have some serious issues like short systemic circulation time or unsatisfactory tissue penetration,33 and extra toxicity induced by excess doses. 5-Amino-N,N′-bis(2,3-dihydroxypropyl)-2,4,6triiodo-1,3-benzenedicarboxamide, one iohexol intermediate (II), has reactive groups. However, II cannot be directly used as a CT contrast agent because of its low solubility in aqueous media. In this work, in order to construct multifunctional PGMA-based polycations for low-toxicity gene vectors and effective water-soluble CT contrast agents, a series of star-like PGMA-based cationic vectors (II-PGEA/II) with abundant flanking polyhydric iodine units were prepared via ATRP and ring-opening reactions (Scheme 1). The five reactive groups of Received: September 12, 2016 Revised: October 4, 2016

A

DOI: 10.1021/acs.bioconjchem.6b00509 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Illustration of the Preparation Processes and Cellular Uptake of PGMA-Based Polycation/pDNA Complexes

area ratio of about 2:1:2 indicated that the intact epoxy rings were kept during the ATRP process. Preparation and Characterization of II-PGEA and IIPGEA/II. For the control II-PGEA group, II-PGMA only reacted with excess EA to give the completely water-soluble polycations (II-PGEA 1 from II-PGMA 1 and II-PGEA 2 from II-PGMA 2). Based on the 1H NMR analysis (Figure 1d), the epoxy groups of II-PGMA were opened completely by excess EA, consistent with our previous work.28 The CT imaging ability of II-containing polycations would be improved with the increase of the II contents. In order to explore the properties of different II percentages, a series of II-PGEA/II polycations with diverse II proportions were prepared. The resultant II-PGEA/II vectors were produced via the ring-opening reactions of the epoxy groups of II-PGMA with II and EA as shown in Table 1. The 1H NMR spectrum was also used to confirm the typical chemical structure of II-PGEA/II (Figure 1e). The peaks at δ = 3.78, 3.49, and 2.63 ppm were the classical marks of the ringopening product which were also found in the spectrum of IIPGEA (Figure 1d). The additional peaks at δ = 8.38, 7.97, 5.45, 4.48−4.76, 3.70, and 3.13−3.41 ppm were obviously associated with II in comparison with the spectrum of pristine II (Figure 1a). Based on the typical 1H NMR spectra of II-PGEA and IIPGEA/II (Figure 1d,e), the epoxy group was completely converted into PGEA or PGEA/II. The UV−vis spectra of different II-PGEA/II polycations were used to quantify the proportion of II in II-PGEA/II (Figure S1; see Supporting Information). The typical absorbance at 320 nm was used to calculate the molar percentage of II. The results were summarized in Table 1. When the percentages of feed II moieties relative to epoxy groups were about 20% and 50%, the different percentages of II in II-PGEA/II were obtained accordingly, i.e., 18.4% for IIPGEA/II 1−1 (from II-PGMA 1), 17.5% for II-PGEA/II 2−1 (from II-PGMA 2), 46.0% for II-PGEA/II 1−2 (from II-PGMA 1), and 48.3% for II-PGEA/II 2−2 (from II-PGMA 2).

II were readily converted into ATRP initiation sites (II−Br5). The resulting II-PGEA/II star vector was composed of one II core and five EA/II-modified PGMA arms. Such amphipathic II-PGEA/II vector readily self-assembles into uniform cationic nanoparticles in aqueous media. The corresponding control IIPGEA vector composed of one II core and five PGEA arms was also synthesized. II-PGEA/II and II-PGEA were systematically compared in detail via a series of transient transfection experiments for the development of CT imaging-guided delivery systems. This work provides a practical method to develop multifunctional polycations by simple ring-opening reaction of aromatic amines in insoluble functional molecules. The present work also would contribute valuable information for the theranostic area.

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RESULTS AND DISCUSSION Preparation and Characterization of II−Br5 and IIPGMA. Star-like II-PGMA composed of one II core and five PGMA arms was prepared via ATRP (Scheme 1). II−Br5 with five initiation sites as the ATRP initiator was first synthesized by esterification of all the reactive hydroxyl and amine groups of II with BIBB. 1H NMR was used to evaluate the chemical structure of II−Br5. As shown in Figure 1a,b, the peaks at δ = 5.51 (2 H), 4.76−4.59 (4 H), 3.87−3.70 (4 H), and 2.02 ppm (30 H) implied that all the hydroxyl and amino groups of II were reacted with BIBB to produce II−Br5 for ATRP. By adjusting the polymerization time from 30 to 45 min, the different molecular weight II-PGMAs were prepared. The number-average molecular weights (Mn) of II-PGMA 1 (from 30 min of ATRP) and II-PGMA 2 (from 45 min of ATRP) were 1.35 × 104 and 2.10 × 104 g/mol, respectively, with the corresponding polydispersity indexes of 1.26 and 1.31. The structure of II-PGMA was also evaluated with 1H NMR (Figure 1c). The peaks at δ = 3.2, 2.8, and 2.6 ppm were clearly attributed to the protons of epoxy rings.28 The corresponding B

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Figure 1. Typical 1H NMR (400 MHz) spectra of (a) II in DMSO-d6, (b) II−Br5 in CDCl3, (c) II-PGMA in CDCl3, (d) II-PGEA in DMSO-d6, and (e) II-PGEA/II in DMSO-d6.

Due to the amphiphilic properties, II-PGEA/II polycations can self-assemble into II-PGEA/II nanoparticles in aqueous media. The particle sizes and zeta potentials of the four types of II-PGEA/II nanoparticles were also summarized in Table 1. The II-PGEA/II nanoparticles demonstrated the particle sizes from 200 to 260 nm. The particle sizes increased with the II

proportion, probably due to the increased steric hindrance induced by hydrophobic II units. The zeta potentials of IIPGEA/II nanoparticles remained positively charged from 32 to 35 mV. To observe the formation of II-PGEA/II nanoparticles, the particle morphologies were imaged by AFM (Figure 2A), where the well-dispersed spherical aggregates appeared. All C

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Bioconjugate Chemistry Table 1. Characterization of II-PGEA/II Polycations sample II-PGEA/II II-PGEA/II II-PGEA/II II-PGEA/II

1−1 1−2 2−1 2−2

molar percentage of II relative to epoxy groupsa (%)

theoretical molar percentage of II relative to epoxy groupsb (%)

18.4 46.0 17.5 48.3

20 50 20 50

particle sizec (nm) 203.6 224.6 239.1 255.2

± ± ± ±

8.6 8.9 11.6 6.6

zeta potentialc (mV) 31.9 33.8 33.6 35.3

± ± ± ±

1.6 0.9 1.3 1.3

a

Calculated from the absorbance of UV−vis spectra at 320 nm. bCalculated from the molar ratios of II/EA in the reactions. cMeasured with the Zetasizer Nano ZS system.

confirmed that pDNA could be effectively compacted by IIPGEA/II. Cytotoxicity Assay. Low cytotoxicity was requested for safe gene carriers in the process of transfection. MTT assay was used to evaluate the cytotoxicity of delivery systems. Figure 4A shows the relative cell viability of all polycation/pDNA complexes. For II-PGEA/pDNA complexes, their cell viability gradually decreased with the increase of N/P ratios. At higher N/P ratios, in addition to compact complexes, excess free polycations also occurred, which induced damages to the cell membranes. However, in comparison with the gold-standard branched PEI (25 kDa)/pDNA complexes, the II-PGEA/ pDNA complexes exhibited much lower cytotoxicity in both HeLa and HepG2 cell lines, probably attributed to the plentiful hydroxyl groups of II-PGEA.26−28 On the other hand, it is very interesting that II-PGEA/II demonstrated lower cytotoxicity than the control II-PGEA counterpart, probably because the introduction of II brought more hydroxyl groups to shield the positive charges. In Vitro Gene Transfection. Plasmid pRL-CMV was used as the report plasmid and delivered by polycations into HeLa and HepG2 cell lines. The transfection efficiency of branched PEI (25 kDa) at its optimal N/P ratio of 10 was chosen as the control.26−28 Figure 4B shows the luciferase transfection results of all II-PGEA or II-PGEA/II/pRL-CMV complexes. For IIPGEA/pRL-CMV complexes, their transfection efficiencies first increased with the increasing N/P ratios until reaching the optimal N/P ratio. The low transfection efficiency at lower N/P ratios was due to the loose compaction of pDNA. Over the optimal N/P ratios, the efficiencies decreased slightly due to the increased cytotoxicity of excess free II-PGEA at higher N/P ratios. On the other hand, although II-PGEA/II possessed improved cell viabilities (Figure 4B), it exhibited poorer transfection performance than the corresponding II-PGEA, indicating that the introduced II did not benefit the resultant transfection process. However, by adjusting the amount of introduced II, at the optimal N/P ratio of 25, the resultant IIPGEA/II 1−1 and II-PGEA/II 2−1 nanoparticles still can produce fairly good transfection performances, which was comparable to the transfection efficiency of PEI (25 kDa) at its optimal N/P ratio of 10. As reported, iodine-based small molecule agents possess excellent CT imaging properties.31,32 Thus, when the amount of introduced II was properly controlled, it is possible to produce II-PGEA/II nanoparticles with satisfactory transfection and CT imaging abilities. Transfection with pEGFP-CD and Antitumor Assay. The suicide gene/prodrug system, particularly 5-fluorocytosine (5-FC)/Escherichia coli cytosine deaminase (ECD), is very promising in treating radio-resistant and chemo-resistant tumors.35 The recombination plasmid encoding enhanced green fluorescent protein-cytosine deaminase (pEGFP-CD) was delivered by the representative II-PGEA 2 and II-PGEA/II

nanoparticles showed fairly good size distribution with the polydispersity index of 0.2−0.3, also indicating the successful formation of the uniform II-PGEA/II nanoparticles. Characterization of Polycation/pDNA Complexes. Agarose gel electrophoresis was used to evaluate the pDNA condensation ability of polycations, where the N/P ratio is defined as the ratio of polycations to pDNA, which could be calculated from the molar ratio of nitrogen (N) in polycation to phosphate (P) in pDNA. As shown in Figure 3A, II-PGEA 1 and II-PGEA 2 completely retarded the migration of pDNA at the N/P ratios of 2.0 and 1.5, respectively. The better condensation ability of II-PGEA 2 is caused by the increased molecular weight. On the other hand, when the suitable II proportion was introduced into II-PGEA/II, no obvious differences in condensation ability were observed. The particle sizes and zeta potentials of polycation/pDNA complexes were shown in Figure 3B. The complexes demonstrated the particle sizes from 140 to 220. The zeta potentials of complexes were positively charged from 22 to 30 mV. With the increase of N/P ratios, all complexes followed decreasing hydrodynamic particle sizes and increasing zeta potentials, which is the typical characteristic of polycation/ pDNA systems.34 In comparison with the pristine II-PGEA/II nanoparticles, all II-PGEA/II/pDNA complexes showed decreased particle sizes and maintained positive zeta potentials, which suggested good compaction of pDNA. The susceptibility of the polycation/pDNA complexes to enzymatic degradation in the presence of nucleases DNase I was also investigated. As shown in Figure 3(c1), the naked pDNA was completely degraded within 30 min. However, pDNA complexed with II-PGEA 2 and II-PGEA/II 2−1 at N/P ratio of 20 showed no significant degradation after 2 h incubation (Figure 3(c2,c3)). These prepared polycations clearly prevented pDNA degradation from DNase I, which secured the proper functions of pDNA during the delivery process. The intracellular release of pDNA caused by polyanion exchange was evaluated by using heparin sodium as a model. As shown in Figure 3D, II-PGEA 2/pDNA and II-PGEA/II 2−1/ pDNA complexes at the N/P ratio of 20 could both release loaded pDNA at proper concentration of heparin sodium. This fact indicated that pDNA in the polycation/pDNA complexes could be released into the cytoplasm for further transfection in present of other polyanions such as proteins. The AFM images of polycation/pDNA complexes at the N/ P ratio of 20 were shown in Figure 2B. The control II-PGEA/ pDNA complexes demonstrated the uniform particles. All the II-PGEA/II/pDNA complexes exhibited smaller sizes than the pristine II-PGEA/II counterparts, consistent with the results of Figure 3B. All the complexes showed similar size distribution to that of the pristine II-PGEA/II nanoparticles. All the results D

DOI: 10.1021/acs.bioconjchem.6b00509 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. AFM images and particle size distribution of (A) pristine II-PGEA/II nanoparticles and (B) polycation/pDNA complexes at the typical N/ P ratio of 20.

2−1 vectors for further study of transfection performance. The EGFP-positive cells were imaged with a fluorescence microscope and quantified by a flow cytometry assay. As shown in Figure 5A, the percentages of the EGFP-positive cells for IIPGEA/II and II-PGEA/II 2−1 at their optimal N/P ratios were 31 ± 1% and 23 ± 3% in HeLa cells (or 26 ± 3% and 19 ± 1% in HepG2 cells), respectively. Such transfection results were

fairly consistent with those of the above luciferase transfection (Figure 4B). For suicide gene delivery systems with 5-FC, CD gene can convert the nontoxic prodrug 5-FC to highly cytotoxic 5fluorouracil (5-FU). pEGFP-CD possesses both sequences of pEGFP and pCD. To evaluate the antitumor effect and determine the suitable prodrug concentration for treatment, various concentrations (0−200 μg/mL) of 5-FC were tested E

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Figure 3. (A) Electrophoretic mobility of pDNA in the complexes of II-PGEA and II-PGEA/II at different N/P ratios; (B) particle sizes (b1) and zeta potentials (b2) of pristine II-PGEA/II nanoparticles and polycation/pDNA complexes; (C) pDNA protection from DNase I enzyme by IIPGEA 2 and II-PGEA/II 2−1 at the N/P ratio of 20 with different incubation time (where naked pDNA was used as a control); and (D) intracellular release of pDNA from II-PGEA 2/pDNA and II-PGEA/II 2−1/pDNA complexes at the N/P ratio of 20 in the presence of heparin.

Figure 4. (A) Cell viability of polycation/pDNA complexes at different N/P ratios in (a1) HeLa and (a2) HepG2 cell lines, and (B) in vitro transfection efficiencies mediated by II-PGEA and II-PGEA/II at different N/P ratios in comparison with branched PEI (25 kDa) at its optimal N/P ratio of 10 in (b1) HeLa and (b2) HepG2 cell lines.

complexes. The cell viabilities for the II-PGEA 2 and II-PGEA/ II 2−1 groups decreased to about 20% at the 5-FC concentration of 100 μg/mL. The relative percentages of dead cells killed with the presence of 5-FC were significantly higher than those of the EGFP-positive cells transfected with IIPGEA 2/pEGFP-CD and II-PGEA/II 2−1/pEGFP-CD. Such a larger difference arose from the favorable bystander effect of 5FC/CD system.36 As a result of CD expression, 5-FC was activated into cytotoxic 5-FU drug in positive cells. On the other hand, the 5-FU produced could freely diffuse across cell membranes to kill adjacent cells. Antitumor abilities were also visualized by cell imaging with PI-staining (Figure 6). The

with the MTT assay as shown in Figure 5B. The control group without complexes maintained high cell viability within the different 5-FC concentrations. After the 24 h treatment at the optimal N/P ratios, both II-PGEA 2/pEGFP-CD and IIPGEA/II 2−1/pEGFP-CD complexes demonstrated decreasing cell viabilities with increasing 5-FC concentrations. Efficient 5-FC concentrations were required for the 5-FC/CD system to effectively kill tumor cells. Due to its better transfection performance, II-PGEA/II 2−1/pEGFP-CD showed lower cell viability than II-PGEA 2/pEGFP-CD. With incubation time extended to 48 h, the excellent results were observed in both cell lines treated with two kinds of F

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Figure 5. (A) Plasmid pEGFP-CD expression mediated by II-PGEA 2 (a1,a3) and II-PGEA/II 2−1 (a2, a4) and (B) cell viability of (b1) HeLa and (b2) HepG2 cells treated with II-PGEA 2/pEGFP-CD and II-PGEA/II 2−1/pEGFP-CD complexes at their optimal N/P ratios under various concentrations of 5-FC.

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CONCLUSIONS We successfully designed and synthesized a series of II-PGEA/ II star polycations with flanking polyhydric iodine groups via ATRP and ring-opening reactions for gene delivery and CT imaging. Due to the hydrophobic property of the introduced II groups, II-PGEA/II polycations can readily self-assemble into micelle-like nanoparticles. The II-PGEA/II nanoparticles possessed good pDNA condensation abilities and exhibited much lower cytotoxicity than PEI (25 kDa). By controlling the suitable amount of introduced II groups, the II-PGEA/II nanoparticles can produce fairly good transfection performances and antitumor effects in different tumor cell lines. More importantly, the II-PGEA/II nanoparticles could induce much better in vitro CT imaging abilities than the commercial CT contrast agent, iohexol. The present design of amphipathic cationic nanoparticles with CT contrast agents would provide a new strategy for the development of multifunctional gene delivery systems.

results of PI-staining cell images were consistent with those of the MTT assay (Figure 5B). After 48 h incubation, massive death of cancer cells occurred. The results further certified the possibility of satisfactory antitumor abilities mediated by IIPGEA/II nanoparticles with the 5-FC/CD suicide gene system. CT Imaging. Iodine-based molecules were widely used as clinical CT contrast agents with promising X-ray attenuation ability and low toxicity. Iohexol, a commonly used commercial CT contrast agent, was chosen as the control. II-PGEA/II 2−1 was tested in aqueous solutions with serial concentrations of iodine from 10 to 50 mM in comparison with iohexol (Figure 7A). A linear curve was obtained by plotting the attenuation intensity as a function of iodine molar concentrations. Under the same molar concentration, II-PGEA/II 2−1 presented a similar imaging capability to the commercial iohexol. The X-ray attenuation ability of the corresponding II-PGEA 2 counterpart was also evaluated (Figure S2; see Supporting Information), where much lower CT values were observed, due to the tiny iodine in II-PGEA 2. Due to the good CT imaging ability of iohexol and II-PGEA/ II 2−1, their in vitro CT imaging assay in tumor cells was performed to simulate the behaviors in organic tissue. The CT images and attenuation values of HeLa and HepG2 cells treated with iohexol and II-PGEA/II 2−1/pDNA complexes (at the N/ P ratio of 25) were investigated in Figure 7B. II-PGEA/II 2−1/ pDNA complex revealed a much better capacity of CT imaging in both cell lines. A reasonable explanation is that the cellular uptake rate of positively charged II-PGEA/II 2−1/pDNA should be higher than that of neutral molecule iohexol. The uptake rates between two different cell lines were responsible for their different CT values.

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EXPERIMENTAL SECTION Materials. Iohexol intermediate (II, 97%), triethylamine (TEA, 99.5%, Extra Dry), and 5-flucytosine (5-FC, 98%) were obtained from Energy-Chemical Co. (China). 2-Bromoisobutyryl bromide (BIBB, 98%), 2,2′-bipyridyl (bpy, 99%), and glycidyl methacrylate (GMA, 95%) were obtained from Tokyo Chemical Industry Co. Ltd. (Japan). Anhydrous dichloromethane (DCM), anhydrous dimethyl sulfoxide (DMSO), copper(I) bromide (CuBr, 99%), heparin sodium, DNase I, ethanolamine (EA, 98%), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), iohexol (95%), and propidium iodinate (PI, 98%) were bought from Sigma-Aldrich G

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Figure 6. PI-staining mediated by II-PGEA 2/pEGFP-CD and II-PGEA/II 2−1/pEGFP-CD at their optimal N/P ratios after 24 and 48 h incubation in (A) HeLa and (B) HepG2 cells with 5-FC at 100 μg/mL.

Figure 7. CT imaging (a1,b1) and CT attenuation values (a2,b2) of commercial agent iohexol and II-PGEA/II 2−1 or II-PGEA/II 2−1/pDNA (at the N/P ratio of 25 for cell treatment) with various iodine molar concentrations: (A) for aqueous solutions and (B) for treated cells.

Escherichia coli and purified according to the supplier’s protocol (Qiagen GmbH, Hilden, Germany). Preparation of II−Br5 Initiators. II (705 mg, 1.0 mmol) and TEA (832 μL, 6 mmol) were added into a 50 mL round flask containing 10 mL of anhydrous DCM. After II was

Chemical Co. (St. Louis, USA). HeLa and HepG2 cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD). Plasmid pRL-CMV encoding Renilla luciferase and plasmid encoding enhanced green fluorescent protein-cytosine deaminase (pEGFP-CD) were amplified in H

DOI: 10.1021/acs.bioconjchem.6b00509 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry thoroughly dissolved, BIBB (742 μL, 6 mmol) in 2 mL of anhydrous DCM was dropped into the aforementioned solution under an ice bath environment for half an hour. The reaction mixture was stirred at room temperature for 24 h and then filtered to discard the precipitates. The organic layer was washed with NaHCO3 solution for three times, treated over Mg2SO4, and dried under reduced pressure to give light brown power, II−Br5 (1.13 g, 78% yield). Preparation of II-PGMA via ATRP. II-PGMA polymers composed of five PGMA arms were preparation under the typical conditions of ATRP. II−Br5 (145 mg, 0.1 mmol, 1 equiv), GMA (2.65 mL, 20 mmol, 200 equiv), and 2,2′bipyridine (156 mg, 1 mmol, 10 equiv) were added into a 50 mL flask containing 5 mL of DMSO. The mixture was degassed by argon for 8 min before adding CuBr (72 mg, 0.5 mmol, 5 equiv). The polymerization was conducted for 30 or 45 min at 30 °C under an argon atmosphere. The mixture was quenched by exposing to air. II-PGMAs were precipitated with 200 mL methanol to remove the catalyst complex and excess monomers. The crude products were reprecipitated cycles with methanol and dried under reduced pressure to produce IIPGMA. The resultant molar weights of II-PGMA 1 from 30 min of ATRP and II-PGMA 2 from 45 min of ATRP were 1.35 × 104 (PDI = 1.26) and 2.10 × 104 g mol−1 (PDI = 1.31), respectively. Preparation of EA/II-Functionalized II-PGMA. The preparation procedures of EA-functionalized II-PGMA (IIPGEA) followed our previous work.26 Briefly, II-PGMA (100 mg), excess EA (1.0 mL), and TEA (0.2 mL) were added in 8 mL of DMSO. The reaction mixture was conducted at 80 °C for 1 h after being degassed by argon for 5 min. The II-PGEA product was purified through dialysis (MWCO 3500) before lyophilization. For the preparation of EA/II-difunctionalized IIPGMA (II-PGEA/II), II-PGMA (100 mg), TEA (0.2 mL), and II/EA reagents with a designed molar ratio [II]:[EA]:[epoxy groups of II-PGMA] of 1:4:1 (or 2.5:2.5:1) were dissolved in 8 mL of DMSO. The reaction mixture was degassed as mentioned earlier by argon for 5 min and stirred at 40 °C for 24 h. Then, the temperature was raised to 80 °C for 40 min to complete the ring-opening reaction. The II-PGEA/II product was purified through dialysis (MWCO 3500) and the insolubles, mainly remaining II species, were removed before lyophilization. Characterization. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker ARX 400 MHz spectrometer using DMSO-d6 or CDCl3 solvent with tetramethylsilane (Me4Si) as an internal standard to determine chemical structures. The molecular weights of II-PGMAs were determined by a Waters 1515 gel permeation chromatography (GPC) system equipped with a Waters Styragel columns and a Waters-2414 refractive index detector. DMSO was used as the eluent with the flow rate of 1.0 mL·min−1, where monodispersed polyethylene glycol (PEG) standards were used. UV−vis spectra were collected by a Shimadzu UV-2600 UV− vis spectrophotometer, where samples were dissolved in DMSO before experiments. The plasmid pRL-CMV and pEGFP-CD were stored at a concentration of 0.1 mg·mL−1. The polycation/pDNA ratio was expressed as the molar N/P ratio of nitrogen (N) in polycation to phosphate (P) in pDNA. All polycation/pDNA complexes were performed by mixing equal volumes of polycation and pDNA solutions to achieve the designed N/P ratios. Each

mixture was vortexed thoroughly before incubation for 30 min at room temperature. The ability of polycations to bind pDNA was assessed by agarose gel electrophoresis as described earlier.27 A Zetasizer Nano ZS system (Malvern Instruments, Southborough, MA) was used to measure the particle size and zeta potential of polycation/pDNA complexes. The morphology of complexes was visualized by an atomic force microscopy (AFM) system with a Dimension Icon model (Bruker, Santa Barbara, CA). The DNase I protection assay followed our previous work.37 The simulate intracellular pDNA release was evaluated as described in the literature.38 Cytotoxicity Assay. The cytotoxicity of polycation/pDNA complexes in HeLa and HepG2 cell lines was evaluated by using MTT assay as described earlier.27 Briefly, the cells were seeded in a 96-well plate at a density of 104 cells per well with 100 μL DMEM and incubated for 24 h in a typical condition. Fresh media containing polycaion/pDNA complexes (3.33 μg/ mL of pDNA) at various N/P ratios were used to replace culture media, and the incubation was kept for another 4 h. The cells were washed with PBS three times before 10 μL of MTT (5 mg/mL in PBS) per well was added to achieve a final MTT concentration of 0.5 mg/mL. After further 4 h of incubation, the excess dye was removed and the cells were washed with PBS three times. Then, 100 μL DMSO was added 100 μL per well to dissolve the produced formazan crystals. A Bio-Rad Model 680 Microplate Reader (UK) was used to measure the absorbance of 570 nm. The cell viability was the average of six parallel wells. In Vitro Transfection Assay. Plasmid pRL-CMV was used as a reporter gene to be performed in HeLa and HepG2 cell lines by the typical procedure.39 The cells were seeded into a 24-well plates with a density of 5 × 104 cells per well in 0.5 mL of media and incubated for 24 h. Then, the media were replaced with 0.3 mL of fresh ones per well and 20 μL of polycation/pDNA complexes at the N/P ratios of 5 to 30 were added to achieve a finial concentration of 3.33 μg/mL of pDNA. After another 4 h, the media were replaced with 0.5 mL of fresh normal ones and the cells were further incubated for 20 h to achieve total 24 h transfection. At the end, PBS was used to wash the cultured cells three times, 100 μL of lysis reagents per well was added to lyse for 2 h. The result of the luciferase gene expression was expressed as relative light units per milligram of cell protein lysate (RLU/mg protein) with a commercial Promega kit and a luminometer (Berthold Lumat LB 9507, Berthold Technologies GmbH. KG, Bad Wildbad, Germany). In Vitro Transfection with pEGFP-CD and Antitumor Effects. MTT assay and cell imaging were used to evaluate the transfection levels of plasmid pEGFP-CD which possessed both sequences of pEGFP and pCD. The EGFP-positive cells were imaged on a Lecia DMI3000B Fluorescence Microscope and quantified by a flow cytometry assay (MoFlo XDP, Beckman, USA).40 The in vitro antitumor effects of the pEGFP-CD/5-FC system were evaluated using similar procedures to those of the pCD/5-FC system as described before.36 Briefly, 104 cells were seeded into a 96-well plate per well and incubated for 24 h. Then, 100 μL of fresh media containing 6.67 μL of polycation/ pDNA complexes (3.33 μg/mL of pDNA) at their optimal N/P ratios were used to replace the old ones. After additional 4 h, the media were replaced with fresh media containing various concentrations of 5-FC and incubated for further 20 or 44 h. After totally 24 or 48 h of transfection, MTT assay procedure I

DOI: 10.1021/acs.bioconjchem.6b00509 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry or PI-staining by adding 10 μL of PI (2 mg of PI dissolved into the mixture of 4 mL of 0.65 M aqueous of D-mannitol and 1 mL of acetone) per well were performed. CT Imaging. CT scans of both aqueous solutions of polycations and cell samples were acquired from a single photoemission computed tomography (SPECT), where all CT values were processed with Amira 4.1.2 software from manufacturer. Various aqueous solutions with the iodine concentrations of 10 to 50 mM (mass concentration of 6 to 30 mg/L) were prepared. The in vitro imaging procedures were similar to the literature.39 In brief, 106 HeLa or HepG2 cells were cultured with 5 mL of media in 25 cm2 cell culture flask for 24 h. Then, 5 mL of media containing amounts of polycation/pDNA at its optimal N/P ratio ([I] = 0, 0.3, 0.6, 0.9, and 1.2 mM) were used to replace the old media. The cells were washed with PBS three times, trypsinized, and centrifuged after 4 h of incubation. Then the cells were resuspended with 0.2 mL of PBS and stored in 0.2 mL tubes for CT imaging. Statistical Analysis. All experiments were repeated at least three times, where the data are presented as means ± standard deviation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00509. UV−vis spectra of II and II-PGEA/II and CT images and values of II-PGEA 2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by NNSFC (National Natural Science Foundation of China, grant numbers 51325304, 51473014, 51503012, and 51521062), Opening Project of State Key Laboratory of Polymer Materials Engineering (Sichuan University) (Grant No. sklpme2014-423), BUCT Fund for Disciplines Construction and Development (Project No. XK1512), Innovation and Promotion Project of Beijing University of Chemical Technology, and Collaborative Innovation Center for Cardiovascular Disorders, Beijing Anzhen Hospital Affiliated to the Capital Medical University. The authors gratefully acknowledge the assistance of CT imaging from Professor Xingjie Liang, National Center for Nanoscience and Technology.

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DOI: 10.1021/acs.bioconjchem.6b00509 Bioconjugate Chem. XXXX, XXX, XXX−XXX