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Galactosylated Poly(2-(2-aminoethyoxy)ethoxy)phosphazene/ DNA Complex Nanoparticles: In Vitro and In Vivo Evaluation for Gene Delivery Yongxin Yang, Zhiwen Zhang, Lingli Chen, Wangwen Gu, and Yaping Li* Center for Drug Delivery System, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China Received November 28, 2009; Revised Manuscript Received March 4, 2010

To achieve efficient gene delivery to the tumor after intravenous administration, biodegradable poly(2-(2aminoethyoxy)ethoxy)phosphazene (PAEP) was modified by lactobionic acid, bearing a galactose group as a targeting ligand. Galactosylated poly(2-(2-aminoethyoxy)ethoxy)phosphazene (Gal-PAEP) with 4.9% substitution degree of galactose could condense pDNA into nanoparticles with a size around 130 nm at the polymer/DNA ratio (N/P) of 2-40. For BEL-7402 cells, the in vitro transfection efficiency of gal-PAEP/DNA complex nanoparticles (gal-PACNs) was much higher than that of the PAEP/DNA complex nanoparticles (PACNs). MTT assay indicated that the cytotoxicity of PACNs significantly decreased after conjugating with the galactose moiety. Gal-PACNs displayed the selective gene expression in the tumor and liver with relatively low gene expression in the lung or other organs compared with PACNs. These results suggested that gal-PACNs could be a promising targeting gene carrier to deliver a therapeutic gene in future.

1. Introduction Receptor-mediated gene delivery systems conjugated with targeting moiety, such as antibody,1 asialoglycoprotein,2 transferrin,3 folate,4 epidermal growth factor (EGF),5 and so on, have been widely investigated to achieve the efficient active gene delivery. It is well-known that the asialoglycoprotein receptor (ASGP-R) is abundantly expressed in normal hepatocytes and hepatocellular carcinoma cell lines, such as HepG2 cells, a human malignant hepatic cell line and parental human hepatocellular carcinoma BEL7402 cells. ASGP-R could selectively bind with galactose and N-acetylgalactosamine residues on small molecules or particles. Galactose as the targeting ligand was because galactose receptors almost exclusively found in the liver, as well as simple coupling chemistry and low immunogenicity compared with antibody or peptide conjugates. Although a few reports have previously been conducted about the in vitro and in vivo behaviors of galactosylated vectors,6-11 few research have been done about their in vivo application for gene delivery in mice bearing tumor, which could be due to the complicated environment of xenografted model of human hepatocellular carcinoma. As a result, it is necessary to investigate in vivo behavior of galactosylated vectors for gene delivery in tumor bearing mice. Polyphosphazenes, which is a relatively novel class of polymers with a long-chain backbone of alternating phosphorus and nitrogen atoms with two polar phosphorus-chlorine bonds, showed good prospect for biomedical application because of its synthetic flexibility and excellent hydrolytic degradability. In particular, the cationic poly(organo)phosphazenes could be prepared by replacing chlorine atoms with amino groups in different charge types for gene delivery.12-15 However, the great efforts have to be made before optimal gene delivery system with high transfection efficiency and low cytotoxicity are found. It has been reported that polymers with primary amino groups are the convenient for coupling with target moiety and the most * To whom correspondence should be addressed. Tel.: +86-21-50806820. Fax: +86-21-5080-6820. E-mail: [email protected].

efficient in transfecting cell lines.16,17 In this work, poly(2-(2aminoethoxy)ethoxy)phosphazene (PAEP) with plenty of primary amino groups was synthesized and modified by galactose moiety to investigate in vitro and in vivo transfection efficiency of cationic polyphosphazenes for targeted DNA delivery into hepatocytes. The gene expression of galactosylated poly(2-(2aminoethyoxy)ethoxy)phosphazene/DNA complex nanoparticles (gal-PACNs) to a distant solid tumor after intravenous administration was investigated, and the cell viability, the cell uptake, and in vitro transfection also were evaluated.

2. Experimental Section 2.1. Materials. 2-(2-Aminoethyoxy)ethanol and polyethylenimine (PEI, 25K) were obtained from Aldrich. Cytochrome C (MW 12327) was purchased from Sigma. Sodium hydride was from Fluka. Lactobionic acid (LA) was purchased from Sinopharm Chemical Reagent Co. Ltd. (China). Trifluoroacetic acid (TFA), di-tert-butyl dicarbonate, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from GL Biochem Ltd. (Shanghai, China). Thiazolyl blue and tetrazolium bromide (MTT) were obtained from Genebase. The pGL2-control vector was purchased from Promega Corp. (Madison, WI) and purified using QIAGEN Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). pGL-2Luc labeled with YOYO-1 (Invitrogen) was used for the evaluation of cellular uptake. Briefly, 200 µL of pGL-2Luc (0.2 µg/mL) was mixed with 10 µL of 50 µM YOYO-1 and incubated at room temperature for 1 h in the dark. FITC-labeled polymers (FITC-PAEP and FITC-gal-PAEP) were prepared as follows: PAEP or gal-PAEP (50 mg) was dissolved in water (5 mL), and then FITC (5 mg) in DMSO was added. After the mixture was stirred at room temperature for 24 h in the dark, the reaction was quenched by adding ammonium chloride to a final concentration of 50 mM for an additional 2 h. Then, the mixture was dialyzed in water using a SeamLess Cellulose Tubing (molecular weight cut off limit ) 5000; Viskase Sales Corp., U.S.A.) and lyophilized. The content of the fluoresceinthiocarbamyl moiety (FTC) of polymers was determined spectrophotometrically by visible absorption at 490 nm with reference

10.1021/bm901346m  2010 American Chemical Society Published on Web 03/21/2010

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Yang et al.

Figure 1. The scheme of the synthesis of gal-PAEP.

to FITC. The content of FTC in FITC-PAEP and FITC-gal-PAEP was 6.7 and 5.6% (w/w), respectively. 2.2. Synthesis and Characterization of gal-PAEP. The polydichlorophosphazene (PDCP) intermediate was obtained as previously described.18 Poly(2-(2-aminoethyoxy)ethoxy)phosphazene (PAEP) was synthesized according to the method described by Allcock.19 The scheme for the synthesis of gal-PAEP was shown in Figure 1. The chemical conjugate of gal-PAEP was formed via an active ester intermediate using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in water. Briefly, the carboxyl group of LA (0.15 g, 10 mol % of the primary amino groups in PAEP) was activated by the NHS/EDC dissolved in water. EDC was a 5-fold molar excess over LA and NHS/EDC molar ratio was 1:1. The activated LA solution was added to the PAEP (0.22 g) solution of 5 mL and allowed to react with stirring for 24 h at room temperature. The reaction mixture was dialyzed using a cellulose membrane (MW cutoff 6000-8000) against distilled water for 3 days. The resulting solution was lyophilized and the composition of galPAEP was determined by NMR spectra. 1H and 31P NMR spectra of polymer were obtained from Varian Mercury Plus-400 NMR spectrometer (Varian, U.S.A.). The chemical shifts were given relative to tetramethsilane or 85% H3PO4 as an external standard. The molecular weight and distribution were determined by gel permeation chromatograph (GPC, Waters 600) with acetonitrile/water/trifluoro acetic acid (10/90/0.05, v/v) as the eluent with flow rate (0.5 mL/min). The calibrations were Cytochrome C standards (Mw 12327). 2.3. Preparation and Characterization of gal-PACNs. Polymer/ DNA complex nanoparticles including PACNs, gal-PACNs, and PEICNs were prepared as follows: the solution containing various amounts of polymer was added to 100 µL of pDNA (20 µg) in distilled water and vortexed for 10 s. The mixture of pDNA and polymer was incubated at room temperature for 15 min. The amount of polymer and pDNA were expressed as N/P ratio. Complex formation was confirmed by electrophoresis on a 1% agarose gel with Tris-acetate (TAE) running buffer at 110 V for 45 min. DNA was visualized with ethidium bromide (0.2 mg/mL). For in vivo experiments, complex nanoparticles were prepared by adding plasmid DNA solution at a concentration of 40 µg/mL to the polymer solution with the concentration of 80 µg/mL for PEI, 700 µg/mL for PAEP, and 700 µg/mL for gal-PAEP, respectively. Size and ζ-potential of complex nanoparticles were measured by laser light scattering following their dilution with water using a Nicomp 380/ZLS ζ-potential analyzer (Santa California, U.S.A.). 2.4. Cell Culture and Cytotoxicity Assay by MTT. Hela human cervix epithelial carcinoma cells, COS7 and HepG2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA)

and parental human hepatocellular carcinoma BEL-7402 cells were obtained from Shanghai Institute of Materia Medica, CAS. Hela cells were incubated in DµLbecco’s modified Eagle medium (Gibco, Carlsbad, CA) and BEL-7402 cells were incubated in RPIM-1640 (Gibco, Carlsbad, CA) supplemented with streptomycine at 40 µg/mL, ampenicillin at 40 U/mL, and 10% fetal bovine serum. The cells were maintained at 37 °C in a 5% CO2 humidified atmosphere. Both the cytotoxicity of gal-PAEP and gal-PACNs were evaluated by MTT assay on BEL-7402 cells. BEL-7402 cells were seeded on a 96-well culture dish at a density of 1 × 104 cells/well and cultivated in complete medium for 24 h. Then, the culture medium was replaced with fresh complete medium containing polymer at different concentrations or polymer/DNA complex nanoparticles at various N/P ratios with subsequently incubated for 3 h. After that, the medium was replaced again prior to the addition of 20 µL/well of MTT solution. Cells were incubated for 4 h at 37 °C in 5% CO2. Then 150 µL of DMSO was added to dissolve the crystals formed by living cells. The absorbance was measured at 490 nm (Bio-Rad Model 550, Hercules, CA). Untreated cells in medium were used as positive reference. Relative cell growth was related to untreated cells and calculated by A(test)/A(control) × 100. 2.5. In Vitro Transfection. For the in vitro transfection experiment, Hela and BEL-7402 cells were seeded into a 24-well plates at a density of 1 × 105 cells per well in 500 µL complete medium 24 h prior to transfection. After the cells were incubated with serial complex nanoparticles at different polymer/DNA ratios in complete medium for 3 h at 37 °C in 5% CO2, the medium was replaced and the cells were further incubated for 45 h. The transfection cells were washed three times with PBS and lysed with 150 µL of gene lysis buffer. The luciferase assay was carried out according to the manusfacture’s instruction (Promega, USA). Briefly, 100 µL of luciferase assay reagent was mixed with 20 µL of the cell lysate and relative light units (RLU) were measured with a chemiluminometer (NOVOSTAR, German) for 10 s at room temperature and corrected with the protein concentration. The protein concentrations were determined with a Commassie Blue Staining Kit (Tiangen, China). For competition assay, BEL-7402 cells were preincubated with galactose (20 mM of final volume) for 15 min, then the cells were incubated with pDNA complex nanoparticles for 3 h. The luciferase activity was determined as described above after 45 h further incubation. 2.6. Cell Uptake. To evaluate cellular uptake efficiency, pGL-2Luc encoding luciferase was used and labeled with YOYO-1. At 24 h prior to uptake experiment, the cells were seeded at a density of 1 × 105 cells/well in 24-well plates. Then, complex nanoparticles containing YOYO-1 labeled plasmid DNA (2.5 µg/well) were added to the cells in fresh complete medium. After incubation for 1 h at 37 °C, the cells

Receptor-Mediated Gene Delivery System were washed with cold PBS twice and harvested by trypsinization. To quench the extracellular fluorescence, the cell suspension was mixed with 25 µL of a 0.4% trypan blue (TB) solution in PBS. The mean fluorescence intensity (MFI) of the cells was measured with a flow cytofluorometer (Becton Dickinson, U.S.A.). MFI of PEICNs was normalized to100. 2.7. Biodistribution. Male BALB/c mice (6 weeks old) were inoculated with 100 µL of phosphate buffered saline (pH 7.4) containing 1.2 × 106 BEL-7402 cells by subcutaneous injection in the left flank. Biodistribution and in vivo transfection experiments started after tumors reached a volume of about 200-300 mm3. For in vivo biodistribution, FITC-labeled PACNs or gal-PACNs solution with N/P ratio of 40 was injected into mice bearing tumor via the tail vein at a dose of 40 µg DNA/mouse. At 15 and 90 min after injection, the mice were anesthetized with ether, then killed by decapitation. The principal organs (including heart, liver, spleen, lung, kidney and tumor) were removed and measured on weight. All the tissues were homogenized in 1 mL of reporter lysis buffer using a glass homogenizer with a Teflon pestle, respectively. Then, the samples were centrifuged at 3000 rpm for 5 min after sufficient mixing. The supernatant was measured at 520 nm, with an excitation at 485 nm using an Infinite 200 spectrofluorometer (Tecan, Austria). 2.8. In Vivo Transfection Experiment. For in vivo transfection experiment, 400 µL of gal-PACNs, PACNs or PEICNs dispersion containing 40 µg pGL2-luc with N/P ratio of 40, 40, and 15 was administered into mice bearing tumor by the tail vein, respectively. At 48 h after injection, the mice were sacrificed by cervical dislocation and the collected organs were washed twice with cold saline, then homogenized with 1 mL lysis buffer using a tissue homogenizer. The resulting tissue homogenates were left on ice for 30 min, vortexed for 20 s, and subsequently centrifuged at 15000 × g for 15 min. Luciferase activity was measured by mixing 100 µL of luciferase assay reagent with 100 µL of supernatant using a chemiluminometer (NOVOSTAR, German). Relative light units (RLU) were measured for 10 s at room temperature. Transfection efficiency was expressed as RLU/organ. Data were corrected for background values in nontreated mice. 2.9. Statistical Analysis. Statistical analyses were performed using a Student’s t-test. The differences were considered significant for p value