Versatile Types of MRI-Visible Cationic Nanoparticles Involving

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Versatile Types of MRI-Visible Cationic Nanoparticles Involving Pullulan Polysaccharides for Multifunctional Gene Carriers Yajun Huang,†,‡,§ Hao Hu,†,‡,§ Rui-Quan Li,†,‡,§ Bingran Yu,*,†,‡,§ and Fu-Jian Xu*,†,‡,§ †

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

S Supporting Information *

ABSTRACT: Owing to the low cytotoxicity and excellent biocompatibility, polysaccharides are good candidates for the development of promising biomaterials. In this paper, a series of magnetic resonance imaging (MRI)-visible cationic polymeric nanoparticles involving liver cell-targeting polysaccharides were flexibly designed for multifunctional gene delivery systems. The pullulan-based vector (PuPGEA) consisting of one liver celltargeting pullulan backbone and ethanolamine-functionalized poly(glycidyl methacrylate) (denoted by BUCT-PGEA) side chains with abundant hydroxyl units and secondary amine was first prepared by atom transfer radical polymerization. The resultant cationic nanoparticles (PuPGEA-GdL or PuPGEAGdW) with MRI functions were produced accordingly by assembling PuPGEA with aminophenylboronic acid-modified Gd-DTPA (GdL) or GdW10O369− (GdW) via the corresponding etherification or electrostatic interaction. The properties of the PuPGEA-GdL and PuPGEA-GdW nanoparticles including pDNA condensation ability, cytotoxicity, gene transfection, cellular uptake, and in vitro and in vivo MRI were characterized in details. Such kinds of cationic nanoparticles exhibited good performances in gene transfection in liver cells. PuPGEA-GdW demonstrated much better MRI abilities. The present design of PuPGEA-based cationic nanoparticles with the liver cell-targeting polysaccharides and MRI contrast agents would shed light on the exploration of tumor-targetable multifunctional gene delivery systems. KEYWORDS: pullulan, PGMA, cationic nanoparticle, gene delivery, MRI

1. INTRODUCTION Gene therapy provides a promising treatment for some widely known diseases, such as cancer, HIV, and diabetes.1−5 Nonviral gene vectors act as a significant role in gene therapy while being faced with the challenges of transfection efficiency, cytotoxicity and targetability.6−9 Various polycations have been proposed for gene delivery, such as polyethylenimine (PEI), poly((2dimethyl amino)ethyl methacrylate), polyamidoamine and poly(2-aminoethyl ethylene phosphate).10−17 In our previous work, it was confirmed that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (denoted by BUCT-PGEA) possessed plentiful hydroxyl units and secondary amine and exhibited effective gene transfection performances.18,19 Natural polysaccharides are reproduceable with excellent biocompatibility and low toxicity. Polysaccharides, including cyclodextrin, pullulan, chitosan and dextran, were widely used in gene delivery fields.20−25 Particularly, we reported one liver celltargeting polysaccharide-based gene vector consisting of nonionic pullulan backbones and disulfide-linked PGEA side chains for highly efficient gene delivery in HepG2 cell lines.26 © XXXX American Chemical Society

Polysaccharides were also quite often used in the design of new contrast agents for cancer diagnosis.27−29 Magnetic resonance imaging (MRI) attracted considerable interest due to its excellent properties such as high spatial resolution and deep tissue imaging.30 Gadolinium ions (Gd3+) are generally chosen as longitudinal relaxation time (T1) contrast agents for their large paramagnetic moment and long electronic relaxation time.30 Magnevist, [Gd-DTPA (DTPA = diethylenetriaminepentacetate acid)]2−, was first used for clinical application as a contrast agent. Until now, more than five Gd chelates, such as Gd-DTPA-BMA, Gd-DOTA, Gd-DO3A, Gd-EOB-DTPA and Gd-BOPTA, for intravenous administration have been widely used.31 However, such low molecular weight MRI contrast agents have significant drawbacks such as, nonspecificity, rapid excretion through the kidneys and possible allergic reactions in the recipients.32 Alternatively, polyoxometalates (POMs, with Received: November 15, 2015 Accepted: January 27, 2016

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DOI: 10.1021/acsami.5b11016 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

2. EXPERIMENTAL SECTION

high molecular weight) can serve as inorganic multidentate ligands for Gd3+, by forming complexes such as [GdW10O36]9−, [Gd(BW11O39)2]15− and [Gd(CuW11O39)2].17,33 Additionally, POMs can assemble with cationic molecules to construct hybrid nanostructures due to their negatively charged surface.34,35 During the past years, several polycation-based MRI contrast agents with Gd3+ chelation were also developed by irreversible covalent bonds.36,37 More recently, one PGEA-based supramolecular gene delivery systems with MRI functions were constructed in our lab by assembling adamantine-modified PGEAs with a Gd3+ ion-containing backbone.38 To construct further multifunctional gene vectors, herein, a series of MRIvisible cationic polymeric nanoparticles involving liver celltargeting pullulans were flexibly designed. Because of its specificity for liver, pullulan, one well-known neutral polysaccharide, was exploited for tumor cell-targeting drug or gene delivery.26,39 Different molecular-weight PuPGEA vectors consisting of pullulan backbones and PGEA side chains were first prepared (Figure 1). Then, the cationic nanoparticles

2.1. Materials. 4-dimethylaminopyridine (DMAP, 98%), 2bromoisobutyryl bromide (BIBB, 98%), glycidyl methacrylate (GMA, 98%), 2,2′-bipyridine (Bipy, 98%), copper(I) bromide (CuBr, 99%), ethanolamine (EA, 98%), triethylamine (TEA, 98%), branched polyethylenimine (PEI, Mw ∼ 25 kDa), diethylene triamine pentacetate acid (DTPA, 98%), 3-aminobenzeneboronic acid hemisulfate salt (APBA, 98%), sodium tungstate dihydrate (98%), gadolinium chloride hexahydrate (99.9%), gadolinium nitrate hexahydrate (99.9%), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI) and inhibitor-removal column were purchased from Sigma-Aldrich (St. Louis, MO). GMA was used after removal of the inhibitors in a ready-to-use disposable inhibitor-removal column. The Hela and HepG2 cell lines were purchased from American Type Culture Collection. The used plasmid DNAs (pDNA) including pRL-CMV and pEGFP-N1 were amplified and purified as described earlier.20 The APBA-functionalized Gd-DTPA (GdL) and Na9GdW10O36·18H2O (GdW) were prepared according to the literature.33,40−42 2.2. Synthesis of Pullulan-based PuPGEA. The bromoisobutyryl-functionalized pullulan (Pullulan-Br) was carried out in the following steps. Pullulan (1.86 g) and DMAP (0.38 g) were successively added into a 100 mL round-bottom flask containing 15 mL of anhydrous DMF with continuous stirring at 37 °C. After pullulan and DMAP were dissolved completely, 5 mL of anhydrous DMF containing 0.18 mL of BIBB was added dropwise into the upper solution in ice bath environment. The reaction was conducted for 24 h under nitrogen and then was precipitated with an excessive diethyl ether. The crude product was purified via dialysis before lyophilization. About 1.21 g of Pullulan-Br was obtained. For the preparation of pullulan-graf t-PGMA (PuPGMA) via atom transfer radical polymerization (ATRP), the molar feed ratio [GMA (2 mL)]:[CuBr]:[Bipy] of 100:1:3 was used at room temperature in 5 mL of DMSO containing 0.2 g of Pullulan-Br. In detail, Pullulan-Br, GMA and Bipy were successively dissolved in 5 mL of DMSO. The reaction mixture was degassed by bubbling nitrogen for 20 min. Finally, CuBr was added into the reaction mixture. The polymerization was conducted from 5 to 20 min at room temperature under a nitrogen atmosphere. The reaction was terminated by exposure to air. The PuPGMAs were precipitated in an excess of methanol to remove the catalyst complex. The crude polymer was dried under reduced pressure after being purified by reprecipitation cycles with methanol. The synthesis procedure of ethanolamine (EA)-functionallized PuPGMA (PuPGEA) followed our previous work.19 Briefly, PuPGMA (200 mg), EA (2 mL) and TEA (0.5 mL) were successively added into 5 mL of DMSO with continuous stirring. After degassed by bubbling nitrogen for 10 min, the reaction mixture was performed at 50 °C for 48 h to produce the PuPGEA. The reaction mixture was precipitated with excess diethyl ether. The crude product was redissolved in 30 mL of deionized water and dialyzed for 48 h, prior to being freeze-dried. 2.3. Conjugation of PuPGEA with GdL or GdW. PuPGEA-GdL was achieved by the conjugation of PuPGEA with GdL, where the diol groups of the pullulan backbone of PuPGEA were reacted via etherification with boronic acid species of GdL.43 Briefly, 50 mg of PuPGEA was completely dissolved in 5 mL of PBS (pH = 7.4). Then, a suitable amount of GdL in 5 mL PBS solution, where the molar feed ratio of diol/boronic acid is 3:1, was added dropwise to the PuPGEA solution. The reaction mixture was processed at 25 °C for 24 h. The produced PuPGEA-GdL was purified using a dialysis membrane (MWCO, 3500 Da) for 72 h prior to lyophilization. PuPGEA-GdW was obtained via electrostatic interaction between PuPGEA and GdW. Briefly, 50 mg of PuPGEA and a suitable amount of GdW powder (where the molar feed ratio of the N/Gd is 90:1) were completely dissolved in 5 mL of ultrapure water, respectively. The GdW solution was added dropwise to the PuPGEA solution. The reaction mixture turned out to be slightly white suspension after stirred overnight. PuPGEA-GdW was purified using a dialysis membrane (MWCO 3500 Da) for 72 h, prior to being freeze-dried.

Figure 1. Synthesis procedures of different pullulan-based cationic nanoparticles.

(PuPGEA-GdL or PuPGEA-GdW) with Gd3+ chelation were prepared by assembling PuPGEA with aminophenylboronic acid-modified Gd-DTPA (GdL) or GdW10O369− (GdW) species via the corresponding etherification or electrostatic interaction. The PuPGEA-GdL and PuPGEA-GdW nanoparticles were investigated and compared systematically through pDNA condensation capability, cytotoxicity, gene transfection ability, cellular uptake and MRI. This work would provide valuable information for the development of new MRI-invisible delivery systems. B


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2.10. In Vivo Experiment. In vivo assay was evaluated on BALB/ C mice. Nude mice were injected with PuPGEA2-GdL/pDNA or PuPGEA2-GdW/pDNA nanoparticles (50 μg of plasmid pRL-CMV, N/P = 25) via tail vein, respectively. For transfection assay, the mice were sacrificed by vivisection after 48 h postinjection. Organs were resected, transferred into liquid nitrogen, triturated and homogenized in lysis buffer (10 mg of organ per 100 μL of lysis buffer). Transfection efficiency was expressed as RLU per g of organ. The in vivo MR imaging assay was evaluated by a 7.0-T MRI instrument (BioSpec 70/ 20 USR 7.0 T Bruker). The MR images and the T1 relaxation times were obtained at the postinjection time of 0, 0.5 and 1 h. 2.11. Statistical Analysis. Every experiment was performed at least three times, where the data are presented as means ± s.d. Statistical significance (*P < 0.05) was evaluated using a Student’s ttest when two groups were compared.

2.4. Preparation of PuPGEA-GdL/pDNA and PuPGEA-GdW/ pDNA. As shown in our earlier work,44 all of the PuPGEA/pDNA, PuPGEA-GdL/pDNA and PuPGEA-GdW/pDNA complexes solutions were prepared in water at an nitrogen concentration of 10 mM. N/P ratios were defined to express the polycation-based vectors to pDNA ratios as the molar ratios of the nitrogen (N) in PuPGEA to the phosphate (P) in pDNA (325 g/mol per phosphate group).38 All the PuPGEA/pDNA, PuPGEA-GdL/pDNA and PuPGEA-GdW/pDNA complexes at various N/P ratios were prepared by mixing polycationbased solution and pDNA solution completely, and stood for 30 min. 2.5. Biophysical Characterization. 1H NMR spectra of polymers were recorded on a Bruker ARX 400 MHz spectrometer by using DMSO-d6 (for PuPGMAs) and D2O (for Pullulan-Br, PuPGEA and APBA-functionalized DTPA) as the solvents. The molecular weights of Pullulan-Br and PuPGMAs were measured on a Waters GPC system (DMSO as the eluent, 1.0 mL min−1) and the molecular weights of PuPGEAs were measured on a YL9100 GPC system (acetic buffer solution as the eluent, 0.5 mL min−1) as described in our work.26 The monodispersed PMMA or PEG standards were used to generate the calibration curves. The GdL, GdW, PuPGEA-GdL and PuPGEA-GdW were characterized by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Scientific iCAP 6000 series). The particle size and ζ-potential of polyplexes were performed by dynamic light scattering with Zetasizer Nano ZS (Malvern Instruments, Southborough, MA, USA). The morphologies of polyplexes were visualized by atomic force microscopy (AFM) with a Nanoscope IIIa controller (Bruker, Santa Barbara, CA, USA). Gel electrophoresis was carried out in a Sub-Cell system (Bio-Rad Laboratories). DNA bands were recorded by a UV transilluminator and BioDco-It imaging system (UVP Inc.).19 2.6. Cell Viability. Hela, cervical cancer cell, and HepG2, liver cancer cell, were used to assess the performance of carriers in vitro. Briefly, 2 × 104 cells per well were seeded in 96-well plates and cultured in DMEM with 10% fetal bovine serum. After 24 h of incubation, the culture medium was removed. 100 μL of fresh medium containing PuPGEA-based complexes (0.33 μg of pDNA per well) was added to each well at different N/P ratios. The detailed procedures were described in our previous work.26 2.7. In Vitro Transfection Assay. The transfection efficiencies of PuPGEA/pDNA, PuPGEA-GdL/pDNA and PuPGEA-GdW/pDNA complexes were first evaluated using plasmid pRL-CMV. The Hela or HepG2 cells were seeded at a density of 5 × 104 cells per well in 24well plates. The complexes (20 μL per well containing 1.0 μg of pDNA) at different N/P ratios were added into the media. The transfection details have been described previously.19 The luciferase gene results were expressed as relative light units per milligram of cell protein lysate. The transfection efficiency of the complexes at their optimal N/P ratios was also analyzed with plasmid pEGFP-N1 by a Leica DMI3000B fluorescence microscope. The EGFP-positive cells were counted by flow cytometry (Beckman Coulter, Brea, CA, USA). 2.8. Cellular Uptake. 5 × 104 Hela or HepG2 cells per well were seeded in 24-well plates. The pDNA (pRL-CMV) was labeled with the fluorescent dye YOYO-1 in advance. After 24 h of incubation, the media containing the PuPGEA-based complexes were added. After 4 h of cellular uptake, the cells were analyzed by flow cytometry (BD LSR II, BD, USA).38 The cultured cells were also imaged by a fluorescence microscope (Leica DMI3000B), where the cells were stained with DAPI for 10 min. 2.9. In Vitro MRI Assay. 5 × 106 cells per well were seeded into culture flask. After 24 h of incubation, the media was replaced with 5 mL of fresh media containing PuPGEA2-GdL/pDNA or PuPGEA2GdW/pDNA complexes ([Gd3+] = 0, 5, 10 and 20 μM). After 4 h, the treated cells were washed with PBS for 3 times, trypsinized and centrifuged on the bottom (about 100 μL) of the 0.2 mL centrifuge tube for MRI imaging. The [Gd3+] concentrations of the control pure PuPGEA2-GdL and PuPGEA2-GdW solutions were about 0, 0.05, 0.10, 0.15 and 0.20 mM in PBS. The MR imaging was evaluated by a 7.0-T MRI instrument (BioSpec 70/20 USR 7.0 T Bruker), and the T1 map-RATE sequence with parameters: TR/TE = 400, 800, 1500, 2500, 4000/7 ms; field of view, 4.0 cm2; matrix, 256 × 256; number of excitations, 2.0; slice thickness = 1 mm; slice gap = 0 mm.

3. RESULTS AND DISCUSSION 3.1. Synthesis of PuPGEA-GdL and PuPGEA-GdW. The synthetic routes of PuPGEA-based cationic species containing Gd3+ are illustrated in Figure 1. PuPGEA was prepared by a three-step procedure. Some hydroxyl groups of pullulan were first functionalized by 2-bromoisobutyryl bromide (BIBB) to produce the ATRP macroinitiator (Pullulan-Br). The representative structure of Pullulan-Br was characterized by 1H NMR spectrum in Supporting Information (Figure S1a). It was estimated that about six initiation sites were introduced on one pullulan backbone. The PuPGMA polymers were obtained via ATRP of GMA. The PuPGMAs with different lengths of PGMA side chain could be obtained by changing ATRP reaction time. The number-average molecular weight (Mn) and polydispersity index (PDI) of Pullulan-Br and PuPGMAs were measured using GPC. As shown in Table 1, in this work, Table 1. Characterization of the Polymers sample a

Pullulan-Br PuPGMA1b PuPGMA2b PuPGEA1c PuPGEA2c

Mn (g/mol)d

PDId

monomer repeat units per side chaine

× × × × ×

1.6 1.5 1.4 1.4 1.3

11 28 14 30

1.8 2.7 4.2 3.5 5.5

4

10 104 104 104 104

a

Pullulan-Br contained about 6 ATRP initiation sites. bPuPGMA was synthesized using a molar feed ratio [GMA (2 mL)]:[CuBr]:[Bipy] of 100:1:3 at room temperature in 5 mL of DMSO containing 0.2 g of Pullulan-Br at different reaction time from 5 to 20 min. cPrepared by functionalizing PuPGMA with excessive ethanolamine (EA). dDetermined from GPC. PDI = weight-average molecular weight/numberaverage molecular weight, or Mw/Mn. eDetermined from the molecular weights of Pullulan-Br and GMA or EA-functionalized GMA.

PuPGMA1 (Mn = 2.7 × 104 g/mol) and PuPGMA2 (Mn = 4.2 × 104 g/mol) were obtained. The total numbers of repeat units per PGMA side chain were 11 (for PuPGMA1) and 28 (for PuPGMA2), respectively. The PuPGEA was prepared by the ring-opening reaction of the primary amine group of EA with the reactive epoxide groups of PuPGMA. Table 1 also summarizes the GPC results of PuPGEA1 from PuPGMA1 and PuPGEA2 from PuPGMA2. Typical 1H NMR spectra of PuPGMA and PuPGEA are shown and analyzed in detail in the Supporting Information (Figure S1b,c). For the preparation of PuPGEA-GdL assemblies, GdL was complexed with PuPGEAs by the etherification between the diol units of the pullulan backbone and the boronic acid groups of GdL. The PuPGEA-GdW assemblies were obtained via electrostatic interaction between cationic PuPGEA and anionic C


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ACS Applied Materials & Interfaces GdW. Based on the ICP-MS assay, the mass fraction of Gd3+ in PuPGEA1-GdL (or PuPGEA1-GdW) and PuPGEA2-GdL (or PuPGEA2-GdW) was 3.12% (or 0.26%) and 1.14% (or 0.41%), respectively. With the increase in molecular weight of PuPGEA, the content of diol groups of the pullulan backbone decreased. The lower diol groups of PuPGEA2 made PuPGEA2-GdL possessed lower amount of Gd3+ than PuPGEA1-GdL. On the other hand, probably due to higher condensabilities of PuPGEA2, PuPGEA2-GdW exhibited larger amount of Gd3+ than PuPGEA1-GdW. The sizes and morphologies of PuPGEA-GdL and PuPGEA1-GdW are discussed in the following sections. 3.2. Biophysical Characterization of PuPGEA-based Complexes. The condensation capability of PuPGEA-based gene vectors was analyzed by agarose gel electrophoresis, dynamic light scattering, ζ-potential measurements and AFM images. Figure 2 shows the gel retardation results of the

Figure 3. (a) Particle size and (b) ζ-potential of the PuPGEA-based complexes at various N/P ratios.

complexes at a N/P ratio of 25 in medium with 10% FBS were approximately 180 and 170 nm during an incubation time of 0−5 h. This result indicated that the complexes could be stable during the transfection process because the cellular uptake of the complexes was within the incubation time of 4 h. The ζpotentials of the pristine PuPGEA-GdL and PuPGEA-GdW assemblies ranged from 25 to 35 mV. The ζ-potential of the pristine PuPGEAs was difficult to measure, as the PuPGEAs could not form nanoparticles. The particle sizes (and ζpotentials) of PuPGEA-GdW/pDNA were smaller (and higher) than PuPGEA/pDNA, probably due to the higher condensation ability of PuPGEA-GdW nanoparticles. In addition, the surface charges of all the PuPGEA-based complexes were highly positive (22−35 mV) (Figure 3b), which would result in good affinities for negatively charged cell surfaces.26 The surface charges of complexes became stable when the N/P ratio reached 25. The morphologies of the nanoparticles were studied by using AFM. Figure 4 shows the typical images of PuPGEA2-GdL, PuPGEA2-GdW, PuPGEA2-GdL/pDNA (at the N/P ratio of 25), and PuPGEA2-GdW/pDNA (at the N/P ratio of 25). The compacted complexes exhibited uniform nanospheres. The particle sizes of PuPGEA2-GdW and PuPGEA2-GdW/pDNA were smaller than those of their PuPGEA2-GdL and PuPGEA2-GdL/pDNA counterparts, which was consistent with the results of particle size measurements (Figure 3a). All the above biophysical properties of PuPGEA-based nanoparticles endow them with different potentials for gene delivery. 3.3. Cell Viability Assay. Relative lower cytotoxicity is essential for a promising gene delivery system. The cytotoxicity of the PuPGEA-based complexes was evaluated in Hela and HepG2 cells at various N/P ratios (Figure 5) using MTT assay. In both cell lines, with the increasing N/P ratios, the relative cell viabilities of all the groups were decreased gradually. That is to say, the cytotoxicity increased with the concentration of the carriers. At the same N/P ratio, the high-molecular-weight

Figure 2. Electrophoretic mobility of pDNA in the polyplexes of (a) PuPGEA1, (a′) PuPGEA2, (b) PuPGEA1-GdL, (b′) PuPGEA2-GdL, (c) PuPGEA1-GdW and (c′) PuPGEA2-GdW at various N/P ratios.

PuPGEA-based vector/pDNA complexes with increasing N/P ratios. All of the gene vectors can effectively condensate pDNA. PuPGEA1 and PuPGEA2 completely retarded the migration of pDNA at the N/P ratios of 2.5 and 2.0, respectively. PuPGEAGdL and PuPGEA-GdW demonstrated the similar condensation capability to their PuPGEA counterparts. Generally, a highmolecular-weight polycation possessed better condensation ability. As the molecular weight increased, PuPGEA2, PuPGEA2-GdL and PuPGEA2-GdW exhibited better pDNA condensation abilities than their corresponding counterparts, PuPGEA1, PuPGEA1-GdL and PuPGEA1-GdW. The above results also indicated that the introduction of the suitable amount of Gd species did not obviously affect the condensation capability of the starting PuPGEA vectors. The particle size and ζ-potential of the PuPGEA-based complexes with different N/P ratios are shown in Figure 3. The particle sizes of the pristine PuPGEA-GdL and PuPGEA-GdW assemblies ranged from 270 to 320 nm. The PuPGEA-GdW nanoparticle exhibited slightly smaller particle sizes than its PuPGEA-GdL counterpart. After complexing pDNA, the PuPGEA-GdL/pDNA and PuPGEA-GdW/pDNA complexes exhibited decreasing sizes. The higher-molecular-weight PuPGEA2-based vectors condensed pDNA into a smaller nanoparticle size than its lower-molecular-weight PuPGEA1based counterparts at the same N/P ratio, which was consistent with their corresponding condensation capability (Figure 2). As shown in Figure S2 (Supporting Information), the particle sizes of the PuPGEA2-GdL/pDNA and PuPGEA2-GdW/pDNA D


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exhibited substantially lower cell cytotoxicity at all N/P ratios, probably benefiting from the good biocompatibility of pullulan backbones and the low cytotoxicity of PGEA species. Our previous work indicated that the plentiful hydroxyl groups of PGEA could shield the deleterious surface charges of the carriers.18 3.4. Gene Transfection Assay. The in vitro gene transfection efficiencies of the PuPGEA-based complexes were investigated in Hela and HepG2 cell lines (Figure 6),

Figure 4. Typical AFM images of (a) PuPGEA2-GdL, (b) PuPGEA2GdL/pDNA (N/P = 25), (c) PuPGEA2-GdW and (d) PuPGEA2GdW/pDNA (N/P = 25).

Figure 6. In vitro gene transfection efficiencies of the gene vector/ pDNA complexes at different N/P ratios in (a) Hela and (b) HepG2 cells in comparison with that of PEI (25 kDa, at the N/P ratio of 10) (mean ± s.d., n = 3, *P < 0.05).

where plasmid pRL-CMV was used as the gene reporter. The profile of luciferase expression of PuPGEA/pDNA, PuPGEAGdL/pDNA and PuPGEA-GdW/pDNA at various N/P ratios was compared with that of “gold standard” PEI (25 kDa) at N/ P ratio of 10.21,26 Generally, the transfection efficiencies of all the PuPGEA-based complexes first increased with increasing N/P ratios at lower N/P ratios, and then decreased slightly. At low N/P ratios, such as the N/P ratio of 10, polycations cannot effectively condense pDNA, and the resultant complexes cannot efficiently pass through the cell membrane easily.44 The PuPGEA-based complexes exhibited the optimal transfection efficiencies at the N/P ratio of 25. At the N/P ratios of 30 and 40, the decreased transfection efficiency was probably caused by the increasing cytotoxicity of the excess free cationic vectors (Figure 5). The transfection efficiency of gene vectors was also dependent on the molecular weights.44 The PuPGEA2-based vectors possessed the increased binding abilities and complex stabilities, likely leading to much higher transfection efficiencies. At most N/P ratios, the gene expression levels mediated by PuPGEA-GdL were comparable to those of the corresponding PuPGEA counterpart. The introduced phenylboronic acid

Figure 5. Relative cell viability of the gene vector/pDNA complexes at various N/P ratios in (a) Hela and (b) HepG2 cells (mean ± s.d., n = 6, *P < 0.05).

PuPGEA2-based vectors exhibited slightly higher cell toxicity. In general, the cytotoxicity of polycations increases with their lengths. Compared with PuPGEA, PuPGEA-GdL and PuPGEA-GdW exhibited the increased cytotoxicity. After the cellular uptake of PuPGEA2-GdL/pDNA and PuPGEA2GdW/pDNA complexes, the complex dissociation within cells would release Gd ligands and increase cytotoxicity. PuPGEAGdW demonstrated a slightly larger cytotoxicity than PuPGEAGdL because of the antitumoral instincts of GdW that is a sandwich-gadolinium POMs.33 However, compared with the control “gold-standard” branched PEI (25 kDa)/pDNA complexes, PuPGEA, PuPGEA-GdL and PuPGEA-GdW E


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cells, which was in agreement with the results of luciferase expression (Figure 6). 3.5. Cellular Internalization. The cellular uptakes of PuPGEA2/pDNA, PuPGEA2-GdL/pDNA and PuPGEA2GdW/pDNA at their optimal N/P ratios were imaged (Figure 8) and evaluated by flow cytometry (Figure 9) in Hela and

groups endowed PuPGEA-GdL with a good affinity to the glycoproteins of cell surfaces.45 On the other hand, PuPGEAGdW exhibited the slightly decreased gene transfection efficiencies, probably because of the introduced cytotoxicity of GdW (Figure 5). Additionally, the transfection efficiencies mediated by PuPGEA-based gene vectors in HepG2 cell lines were significantly higher than those in Hela cell lines. In HepG2 cell lines, the transfection efficiencies mediated by PuPGEA, PuPGEA-GdL and PuPGEA-GdW at higher N/P ratios are higher than that of PEI at its optimal N/P ratio of 10. It was well-known that pullulan possessed the liver targeting performance.46−48 As confirmed in our earlier work,26 the pullulan-based gene vectors possessed the good liver celltargeting ability. The transfection performances of gene vectors were also imaged by fluorescence microscopy. The typical images of EGFP expression mediated by PuPGEA2, PuPGEA2-GdL, PuPGEA2-GdW and PEI at their respective optimal N/P ratios are shown in Figure 7. The percentages of the EGFP-positive

Figure 9. Flow cytometry analysis plots by (a) Hela and (b) HepG2 cells treated with PuPGEA2/pDNA, PuPGEA2-GdL/pDNA and PuPGEA2-GdW/pDNA complexes for 4 h at the N/P ratio of 25.

HepG2 cell lines, where plasmid pRL-CMV was labeled by YOYO-1. As shown in Figure 8, YOYO1-labeled pDNA was shown in green fluorescence and the nuclei stained with DAPI were shown in blue fluorescence. More green accumulation was observed in the HepG2 cells. The percentages of YOYO1positive cells were counted by flow cytometry. The results of the PuPGEA2/pDNA, PuPGEA2-GdL/pDNA and PuPGEA2GdW/pDNA complexes were 69.4%, 58.9% and 65.2% in HepG2 cell lines, respectively, higher than those (∼50%) in Hela cell lines (Figure 9). The above results indicated that the

Figure 7. Representative images of EGFP expression in (a) Hela and (b) HepG2 cells mediated by PuPGEA2, PuPGEA2-GdL, PuPGEA2GdW and PEI at their respective optimal N/P ratios.

cells for PuPGEA2, PuPGEA2-GdL, PuPGEA2-GdW, and PEI in Hela (or HepG2) cells are 33.5% (or 42.3%), 35.7% (or 43.7%), 29.7% (or 38.8%) and 17.4% (or 20.1%), respectively. The above suggested that the PuPGEA-based complexes possessed better transfection performances in the HepG2

Figure 8. Fluorescent images of (a) Hela and (b) HepG2 cells treated with PuPGEA2/pDNA, PuPGEA2-GdL/pDNA and PuPGEA2-GdW/pDNA polyplexes for 4 h at the N/P ratio of 25, where the YOYO-1-labeled pDNA are shown in green fluorescence, and the DAPI-labeled nuclei are shown in blue fluorescence. F


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ACS Applied Materials & Interfaces pullulan backbone probably benefited the liver cell uptake (Figure 8). Such enhanced cellular uptake may favor the resultant gene transfection process. 3.6. In Vitro MRI Assay. Gd3+ has been recommended as a promising T1-weighted MRI contrast agents for high efficiency of longitudinal relaxation.30 The T1 relaxation properties of PuPGEA2-GdL and PuPGEA2-GdW in PBS solutions were first investigated (Supporting Information, Figure S3). The T1weighted MR images for the cell-free solutions of PuPGEA2GdL and PuPGEA2-GdW varied markedly, and the signal intensity was positively correlated with the Gd concentration. The linear curves were obtained by plotting the inverse T1 as a function of molar concentration of Gd3+ (Figure S3, top). The brightness of MR images increased with the increasing molar concentration of Gd3+ (Figure S3, bottom). The T1 relaxation properties of Hela and HepG2 cells treated with PuPGEA2-GdW/pDNA and PuPGEA2-GdL/pDNA were subsequently investigated (Figure 10). The MR images of

Figure 11. (a) T1-weighted MR images and (b) inverse T1 values of different organs of the nude mice under preinjection and postinjection of PuPGEA2-GdL/pDNA and PuPGEA2-GdW/pDNA (0.6 μmol Gd kg−1) at different time, where the different white dotted frames denote liver, kidney and spleen.

concentration and the complicated biochemical environment in vivo. The in vivo transfection results in different organs are shown in Figure S5 (see the Supporting Information). The gene transfection efficiencies of PuPGEA2-GdL/pDNA and PuPGEA2-GdW/pDNA in liver were much higher than that of PEI, probably due to the liver cell-targetability of pullulan.26 Moreover, the in vivo MRI imaging abilities of PuPGEA-based complexes were most excellent in liver, where the good in vivo gene transfection efficiency was also produced. The above indicated that the PuPGEA-GdW/pDNA and PuPGEA-GdL/ pDNA complexes remained stable during the transfection process.

Figure 10. (a) Linear fitting of the inverse T1 and (b) T1-weighted MR images of HepG2 and Hela cells treated with PuPGEA2-GdL/pDNA and PuPGEA2-GdW/pDNA at the N/P ratio of 25 under media with different Gd3+ concentrations.

HepG2 cells treated with PuPGEA2-GdW/pDNA or PuPGEA2-GdL/pDNA were apparently brighter than those of Hela cells. This difference seems to be associated with their cellularuptake profiles (Figures 8 and 9). In addition, PuPGEA2-GdW demonstrated the much higher inverse T1 values than PuPGEA2-GdL (GdL, i.e., modified Gd-DTPA) particularly in HepG2 cell lines. The earlier work indicated that GdW is a promising liver-specific MRI contrast agent and displayed a higher relaxivity than that of Gd-DTPA.49 3.7. In Vivo Assay. The PuPGEA2-GdL/pDNA and PuPGEA2-GdW/pDNA complexes were injected via tail vein of the nude mice. The T1-weight MRI assay was performed on the nude mice. Figure 11a shows that the color coded T1weighted MR images of different organs treated by PuPGEA2GdW/pDNA were brighter than those of PuPGEA2-GdL/ pDNA. The T1-weighted MR images of different organs were also provided (Figure S4, Supporting Information). In addition, Figure 11b shows that PuPGEA2-GdL and PuPGEA2-GdW exhibited gradual increases of inverse T1 values within 1 h in liver, kidney and spleen. With the longer circulation, higher amounts of PuPGEA2-GdL/pDNA and PuPGEA2-GdW/ pDNA accumulated in the organs. The differences of MR images and inverse T1 values between PuPGEA2-GdW/pDNA and PuPGEA2-GdL/pDNA are fairly in agreement with the results of in vitro MRI assay (Figure 10). The 1/T1 of PuPGEA2-GdW/pDNA was not significantly higher than that of PuPGEA2-GdL/pDNA probably owing to the low Gd3+

4. CONCLUSION A series of liver cell-targeting PuPGEA-based cationic assemblies with MRI function were successfully synthesized by assembling PuPGEA with GdL or GdW via the corresponding etherification or electrostatic interaction. The resultant PuPGEA-GdL and PuPGEA-GdW nanoparticles had good DNA condensation abilities and exhibited much lower cytotoxicity than PEI (25 kDa). Such kinds of PuPGEA-based nanoparticles exhibited better performances in gene transfection and cellular uptake in hepatocellular liver carcinoma cell lines. PuPGEA-GdL and PuPGEA-GdW demonstrated different MRI properties in vitro and in vivo. PuPGEA-GdW produced better MRI abilities. The present cationic nanoparticles consisting of pullulan, PGEA and MRI contrast agents would favor the development of targetable and visible multifunctional delivery systems.

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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/acsami.5b11016. Details and additional characterization such as 1H NMR assay, complexes stability in serum, and images with PuPGEA-GdL and PuPGEA-GdW and in vivo transfection (PDF). G


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Fu-Jian Xu). *E-mail: [email protected] (Bingran Yu). 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 51221002, 51325304, 51473014 and 51503012), Beijing Natural Science Foundation (project no. 7151002), BUCT Fund for Disciplines Construction and Development, Opening Project of State Key Laboratory of Polymer Materials Engineering (Sichuan University) (Grant No. sklpme2014-423, sklpme2015-4-23), Fundamental Research Funds for the Central Universities (ZY1527), 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 animal experiment from Yang Hu.

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