Urocanic Acid Improves Transfection Efficiency of Polyphosphazene

Feb 2, 2010 - The biodegradable cationic poly(2-(2-aminoethoxy)ethoxy)phosphazene (PAEP) bearing primary amino groups and a new PAEP derivative, ...
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Bioconjugate Chem. 2010, 21, 419–426

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Urocanic Acid Improves Transfection Efficiency of Polyphosphazene with Primary Amino Groups for Gene Delivery Yongxin Yang, Zhiwen Zhang, Lingli Chen, Wangwen Gu, and Yaping Li* Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. Received June 17, 2009; Revised Manuscript Received November 17, 2009

The biodegradable cationic poly(2-(2-aminoethoxy)ethoxy)phosphazene (PAEP) bearing primary amino groups and a new PAEP derivative, urocanic acid (UA) modified PAEP (UA-PAEP), were synthesized and investigated for gene delivery. The results indicated that PAEP was able to condense DNA into complex nanoparticles with the size around 120 nm at the polymer/DNA ratio (N/P) of 35, at which PAEP/DNA complex nanoparticles (PACNs) showed efficient transfection activity in complete medium. After conjugating with UA at the substitution degree of 7% (UA-PAEP7), UA-PAEP7/DNA complex nanoparticles (UP7CNs) exhibited higher transfection efficiency than PACNs and UA-PAEP25/DNA complex nanoparticles (UP25CNs) and much lower cytotoxicity compared with PEI/DNA complex nanoparticles (PEICNs). The transfection experiment using a proton pump inhibitor suggested that the gene expression of PACNs and UP-PAEP/DNA complex nanoparticles (UPCNs) was dependent on the endosomal acidification process. The acetate solution (20 mM, pH5.7) improved the transfection activity of UP7CNs in HeLa and COS 7 cell lines, which was almost comparable to PEICNs at the N/P ratio of 35. Therefore, the results suggested that UP7CNs could be a promising carrier for gene delivery.

INTRODUCTION A number of cationic polymers such as polyethylenimine (PEI) (1), chitosan (2), poly(L-lysine) (PLL) (3), and polyamidoamine dentrimers (4), have been widely investigated for gene delivery due to their obvious advantages over viral gene vectors such as low safety risks, preparation in large quantities easily, and low costs. Among these cationic polymers, PEI has become a golden standard of nonviral gene delivery systems (5), but the high cytotoxicity and nonbiodegradability limit its usage. So far, great efforts have been made to design and develop novel biodegradable polymers with high transfection activity and low cytotoxicity for gene delivery (6, 7). Unfortunately, the ideal cationic polymer has not been found yet. Polyphosphazenes have been investigated for biomedical and pharmaceutical applications such as drug delivery systems (8) and protein matrices (9) because of their synthetic flexibility, excellent hydrolytic degradability, and nontoxic degradable products. For gene delivery, the cationic polyphosphazenes have shown good activity in gene transfection recently (10-12). However, more efforts have to be made before polyphosphazene derivatives with the optimal gene transfer efficiency are found. In our previous work (13), poly(imidazole/DMAEA)phosphazene (PIDP) bearing ternary amino groups showed the ability to condense DNA into complex nanoparticles with a size around 100 nm, which could mediate more efficient transfection than PEI/DNA complex nanoparticles (PEICNs) and poly(di-DMAEA)phosphazene/DNA complex nanoparticles at the same polymer/ DNA ratio (w/w) in serum-free medium. However, PIDP/DNA complex nanoparticles exhibited much lower gene expression level in medium containing 10% FBS. In order to meet the requisites of in vivo gene delivery, we are looking for a novel cationic polyphosphazene derivative that could achieve efficient transfection in the presence of serum. * Corresponding author. Address: 555 Zuchongzhi Road, Shanghai 201203 China. Tel.: +86-21-5080-6820. Fax: +86-21-5080-6820. E-mail address: [email protected].

On the cellular level, the complex nanoparticles must overcome many obstacles before they release nucleic acid at the target site of action. The first obstacle is traversing the cellular membrane. Complex nanoparticles are able to pass the cellular membrane via endocytosis as long as they have the suitable surface properties such as the appropriate size range and the positive charges (14, 15). However, endocytotic uptake may not only expose complex nanoparticles to enzymatic degradation in the lysosome, but also entrap and never release them. To circumvent above difficulties, we synthesized biodegradable poly(2-(2-aminoethyoxy)ethoxy)phosphazene (PAEP) bearing primary amino groups for efficient gene delivery first. Considering urocanic acid (UA) with an imidazole ring for enhanced endosomal escape, a new PAEP derivative, UA modified PAEP (UA-PAEP), was synthesized in an attempt to improve the transfection activity. In this work, the buffering capacity of polymers, the transfection activity of complex nanoparticles in the presence of serum, and the influence of UA substitution degree on the transfection efficiency were investigated. In addition, the physicochemical property and the cell uptake of the polymer/DNA complex nanoparticles were characterized for further exploration of the mechanism of in vitro gene transfection.

EXPERIMENTAL PROCEDURES All the reagents (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China), unless otherwise noted. NH4Cl was dried in a desiccator over P2O5 (Panreac). Tetrahydrofuran (THF) was treated with KOH and distilled twice from Na in the presence of benzophenone. Petroleum ether refers to that fraction with a boiling point in the range 60-90 °C. PCl5 was purified by sublimation. Sulfamic acid (HSO3NH2), CaSO4 · 2H2O, 2-(2-aminoethyoxy)ethanol (Aldrich), sodium hydride (Fluka), and 1,2,4-trichlorobenzene were used as purchased. Trifluoroacetic acid (TFA), N,N′dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS) were purchased from GL Biochem (Shanghai) Ltd.

10.1021/bc900267g  2010 American Chemical Society Published on Web 02/02/2010

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Figure 1. Synthetic scheme of poly(2-(2-aminoethyoxy)ethoxy) phosphazene(PAEP) and UA-PAEP.

Polyethylenimine (PEI 25K), poly(L-lysine) (PLL), and UA were obtained from Aldrich. YOYO-1 was obtained from Invitrogen and Trypan Blue from TianGen. Bafilomycin A1 was purchased from Sigma. pEGFP-N1 (4.7 kb) encoding green fluorescent protein driven by immediate early promoter of CMV was purchased from Clontech Laboratories (Palo Alto, CA, USA). pGL-2Luc was purchased from Promega Corp. (Madison, WI, USA). The plasmid DNA (pDNA) was amplified in DH5R strain of E. coli and purified by EndFree Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). Cell Lines. Hela cells and COS 7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in DMEM containing 10% fetal bovine serum (FBS), streptomycin at 40 µg/mL, and ampicillin at 40 U/mL. Cells were maintained at 37 °C in a humidified and 5% CO2 incubator. Synthesis of PAEP and UA-PAEP. Poly(dichloro)phosphazene was prepared directly from PCl5 and NH4Cl as described in our previous work (13). The synthesis of PAEP (Figure 1) was carried out according to the Allcock’s procedure (16). To synthesize UA-PAEP, the carboxyl group of UA was activated by the NHS/DCC dissolved in dimethylformamide (5 mL). DCC was 1.2-fold molar excess over UA and NHS/DCC molar ratio was 1:1. The activated UA solution was added to PAEP solution and triethylamine in methanol (10 mL), and the resultant solution was stirred for one day at room temperature. The reaction mixture was dialyzed using cellulose membrane (MW cutoff 6000-8000) against distilled water for 3 days. The resulting solution was lyophilized, and the composition of UAPAEP was determined by NMR spectra. 1H NMR and 31P NMR spectra of polymer were obtained from Varian Mercury Plus400 NMR spectrometer (Varian, USA). 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 chromatography (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 peptide standards. Buffering Capacity. The buffering capacity of PAEP, UAPAEP, PLL, and PEI were measured by titration according to the method described by Tseng (17). The polymers were diluted to a final concentration of 0.3 mg/mL with 0.1 N NaCl. The solution was adjusted to pH 7.5 before titration proceeded. An aliquot of 0.1 N HCl was successively added into 30 mL of

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polymer solution (0.3 mg/mL), and the change in pH was monitored by a pH meter (Sartorius, Germany). Preparation and Characteristics of Polymer/DNA Complex Nanoparticles. The polymer/DNA complex nanoparticles were prepared by adding 100 µL polymer solution with various amounts of polymer to 100 µL of pEGFP (20 µg) in distilled water or 20 mM acetate (pH 5.7) and vortexed for 10 s. The resulting complex nanoparticles were allowed to sit at room temperature for 30 min, then confirmed by electrophoresis on a 1% agarose gel with Tris-acetate-EDTA buffer system (pH 8.0) at 110 V/cm for 45 min. DNA was visualized using ethidium bromide staining. The polymer/DNA charge ratio (N/ P) was defined as the ratio between the moles of the amine groups of PAEP or UA-PAEP to those of the phosphate ones of DNA. For plasmid DNA, 330 g/mol corresponds with the average mass of a repeating unit bearing one negative phosphate group. Size and ζ-potential of complex nanoparticles were measured by laser light scattering following their dilution with water or 20 mM acetate (pH 5.7) using a Nicomp 380/ZLS zeta potential analyzer (Santa California, USA) at 25 °C. For size measurement, scattered light was detected at a 90° angle and the instrument was calibrated with an aqueous poly(styrene) dispersion of particles of 100 nm. ζ-potential measurements of the complexes were carried out in the standard capillary electrophoresis cell at 25 °C. In Vitro Transfection Experiment. In vitro transfection efficiency was evaluated on Hela and COS 7 cell lines using a plasmid containing a reporter gene encoding enhanced green fluorescence protein (EGFP). Cells were seeded into a 24-well plates at a density of 1 × 105 cells per well in 500 µL of complete medium 24 h prior to transfection. After the polymer/ DNA complex nanoparticles containing 2.5 µg of DNA were incubated with cells at 37 °C for 4 h, the medium was replaced with fresh complete medium. After 48 h, the cells were washed twice with PBS and collected. The fluorescence intensity of positive cells was measured with a flow cytofluorometer (Becton Dickinson, USA) equipped with an argon ion laser. PEICNs were used as positive control. The transfection experiment involving bafilomycin A1 was performed as described above except that the cells were preincubated with 200 nM bafilomycin A1 at 37 °C for 30 min. Cell Viability Assays. The cytotoxicity of complex nanoparticles was determined by MTT assay. The cells were plated on a 96-well culture dish at a density of 2 × 104 cells/well and cultivated in complete medium. After 24 h, the culture medium was replaced with fresh complete medium containing complex nanoparticles at different polymer/DNA (N/P) ratio. After 4 h, the incubation medium was replaced again with fresh medium and incubation for an additional 42 h. The medium was removed and MTT solution (5 mg/mL) was added. Cells were incubated for 4 h at 37 °C in 5% CO2. Then, the medium containing MTT was removed, and 150 µL of DMSO was added to dissolve the crystals formed by living cells. The absorbance was measured at test wavelengths of 490 nm (Bio-Rad model 550, Hercules, CA, USA). Cell Uptake of Polymer/DNA Complex Nanoparticles. To evaluate cellular uptake efficiency and subsequent intracellular routing of complex nanoparticles, pGL-2Luc encoding luciferase was used and labeled with YOYO-1. Briefly, 200 µL of pGL2Luc (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. At 24 h prior to uptake experiment, the cells were seeded at a density of 1 × 105 cells/well in 24-well microtiter plates. Then, the complex nanoparticles containing 2.5 µg of YOYO-1-labeled plasmid DNA were added to the cells in fresh complete DMEM medium. After incubation for 1 h at 37 °C, the cells were washed

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Figure 2. 31P NMR spectra of PAEP (a); 1H NMR spectra of polymer 1 (b), PAEP (c), and UA-PAEP (d).

with cold PBS twice. Fluorescent microscope images of cells were taken simultaneously using an Oliympus Inverted microscope (Japan). Finally, the cells were 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. MFI of PEICNs was normalized to 100. The uptake mechanism of PACNs and UPCNs in Hela cells was examined by means of specific inhibitors of the different endocytic pathways. For inhibition experiments, the cells were first incubated with one of the following inhibitors: 56 µM chlorpromazine (to inhibit clathrin-mediated endocytosis), 10 µM nystatin (to inhibit microtubule-mediated endocytosis), 3 µM cytochalasin D (to inhibit macropinocytosis), or 200 µM genistein (an inhibitor of caveolae-mediated endocytosis) for 30 min in completed medium prior to addition of complex nanoparticles to the cells. Inhibitors were used at concentrations in which they were shown to be active but not cytotoxic in COS 7 cells (18). Statistical Analysis. Statistical analysis was performed using a Student’s t-test. The differences were considered significant for p < 0.05 and p < 0.01 indicative of a very significant difference.

RESULTS AND DISCUSSION Synthesis of PAEP And UA-PAEP. Prior to the substitution reaction, one of the two nucleophilic groups in 2-(aminoethoxy)ethanol had to be protected lest cross-linking of the polymer occurred. In this work, the tert-butoxycarbonyl (Boc) group, which decomposes readily by treatment with acid and

remains intact under basic conditions, was used to protect amine groups of 2-(aminoethoxy)ethanol. Once polymer 1 was obtained, the deprotection of tert-butoxycarbonyl group was carried out by trifluoroacetic acid. Since the deprotection reaction was carried out under acidic condition, chain cleavage was considered to be a possible side reaction. However, no signal that could be attributed to decomposition was observed in 31P NMR of PAEP (Figure 2a). A strong single peak of 1H NMR for Boc group, as shown at 1.36 ppm (Figure 2b), disappeared in 1H NMR spectra of PAEP (Figure 2c). The molecular weight of PAEP was 1.47 × 104 Da. To facilitate the endosome escape, PAEP was coupled with UA using an active ester method to obtain UA-PAEP. PAEP was first dissolved in methanol because it was not soluble in aprotic solvents and diluted with dimethylformamide to react with activated UA. 1H NMR of UA-PAEP in D2O was shown in Figure 2d. The substitution degrees of UA were 7% (UA-PAEP7) and 25% (UA-PAEP25) of the total amine functions, respectively. Buffering Capacity of Polymers. The buffering capacity of polymer was tested via titration with 0.1 N HCl. Figure 3 showed that the buffering capacities of UA-PAEP7 and UAPAEP25 were higher than that of PAEP in the range of pH 4-6.6, which indicated that the involvement of UA improved the buffering capacity of PAEP. The buffering capacity of UAPAEP slightly increased when the substitution value of UA increased from 7% to 25%. As positive control, PEI showed excellent buffering capacity. PLL exhibited much lower buffering capacity than other polymers as a negative control. It is believed that the correlation of the pH range to the transfection process is between pH 5 and pH 7. In this work, the range of pH 4-6.6 was chosen to compare the buffering capacities of

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Yang et al. Table 1. Average Particle Size and ζ Potential of Polymer/DNA Nanoparticles Prepared in Different Media at the Polymer/DNA Ratio (N/P) of 35 distilled water

Figure 3. Titration curve of PAEP, UA-PAEP7, UA-PAEP25, and PEI solution (0.3 mg/mL). The polymer solution was titrated with 0.1 N HCl.

Figure 4. DNA retardation assay by agarose gel electrophoresis using 1% agarose in Tris-acetate running buffer (pH 8.0). (A) Lane 1, DNA control; lanes 2-7, PAEP/DNA (N/P) ) 1.2, 2.5, 5, 7.5, 25, and 37, respectively. (B) Lane 1, DNA control; lanes 2-7, UA-PAEP7/DNA (N/P) ) 1.2, 2.5, 5, 7.5, 25, and 37, respectively. (C) Lane 1, DNA control; lanes 2-7, UA-PAEP25/DNA (N/P) ) 1.2, 2.5, 5, 6.7, 7.5, and 25, respectively.

the polymers according to ref 19. On the other hands, UP7CNs showed obvious difference in their buffering effects compared with PAEP in the pH range 4-6.6 rather than in the pH range 5-7. Characterization of Polymer/DNAComplex Nanoparticles. The formation of polymer/DNA complex nanoparticles was confirmed by gel retardation assay. The ratio of polymer/DNA (N/P) at which the electrophoretic mobility of DNA was completely retarded was 2.5:1 for PAEP, 5:1 for UA-PAEP7, and 6.7:1 for UA-PAEP25, respectively (Figure 4). The results indicated that the ability of UA-PAEP condensing DNA was less efficient than PAEP due to the decreased positive charges. It has been reported that the suitable surface property and size range enable the cationic polymer/DNA complex nanoparticles to pass the cellular membrane via endocytosis (14). PAEP could condense DNA into nanoparticles with an average diameter around 120 nm at the polymer/DNA (N/P) ratio from 2.5 to 35 in distilled water. According to transfection experiment (see following section), the polymer/DNA complex nanoparticles prepared either in water or in 20 mM acetate (pH 5.7) at the N/P ratio of 35 were used for following experiments. Table 1 showed that the particle sizes of all complex nanoparticles prepared in water were about 120 nm, which were much smaller than those prepared in 20 mM acetate (pH 5.7). Meanwhile, the surface charges of the above complex nanoparticles prepared

20 mM acetate (pH 5.7)

polymer

particle size (nm)

ζ-potential (mV)

particle size (nm)

PAEP UA-PAEP7 UA-PAEP25

119.4 ( 5.5 1230.6 ( 6.2 115.9 ( 4.3

30.5 ( 6.1 23.9 ( 4.8 6.7 ( 3.3

161.4 ( 5.2 168.8 ( 6.6 145.7 ( 5.9

in distilled water decreased with increasing degree of UA substitution. The ζ potential of PACNs was the highest (30.5 mV), and that of UA-PAEP25/DNA complex nanoparticles (UP25CNs) was only 6.7 mV. Apparently, the conjugation of UA with the amino groups in PAEP decreased the DNA binding ability of polymer, as well as the charge density of complex nanoparticles. In VitroTransfection Efficiency. In order to know the transfection activity of PACNs, the experiment was first performed in Hela cells using pEGFP in serum-free medium. As shown in Figure 5A, PACNs yielded the highest transfection efficiency at the N/P ratio of 7.5, which was almost the same order of magnitude as PEICNs at the optimal ratio (N/P ) 35). This behavior of PAEP was so different from that of another structurally similar polyphosphazene derivative, poly(di-DMAEA)phosphazene (11), which showed much lower transfection efficiency compared with the PEI polyplexes. PAEP and poly(diDMAEA)phosphazene are two cationic polyphosphazenes with an identical backbone containing different side chains, which are primary and tertiary amino groups, respectively. It has been reported that the type of charge group is one of the most important parameters for efficient gene transfer (20-22). Among all carriers with various types of charge groups, polymers with primary amino groups were found to be most efficient in transfecting cell lines (23, 24). So, the different types of charge groups might bring about the variance of transfection level between PAPE and poly(di-DMAEA)phosphazene. Then, in vitro transfection efficiency of PACNs prepared in distilled water was evaluated in Hela and COS 7 cell lines to look for the optimal condition of transfection in complete medium. Figure 5B showed that PACNs mediated the highest level of gene expression at the N/P ratio of 35 in both Hela and COS 7 cell lines. The increase of the optimal transfection ratio (N/P) of PACNs from 7.5 in serum-free medium to 35 in complete medium might result from the decreased positive charges of complex nanoparticles after interaction with the components of serum. It was reported that 20 mM acetate (pH 5.7) could ensure a higher positive charge density (11). So, the transfection efficiencies of PACNs, UPCNs, and PEICNs prepared either in distilled water or in 20 mM acetate (pH 5.7) at the N/P ratio of 35 were investigated to understand the relationship between preparation medium and transfection activity. The results showed that UP7CNs exhibited higher transfection activity compared with PACNs or UP25CNs in both media (Figure 5C) and the transfection level of UP7CNs was almost comparable to that of PEICNs. Despite the slightly lower buffering effect compared to that of UA-PAEP25, UP7CNs showed higher transfection efficiency than UP25CNs. The superior transfection activity of UP7CNs could result from its suitable positive charges, which are helpful for endocytosis, stability in cytoplasm, and dissociation of pDNA from UP7CNs. Besides, improved buffering capacity of UA-PAEP7 could be another reason accounting for the higher level of gene expression of UP7CNs than that of PACNs. With higher buffering capacity than UA-PAEP7, UP25CNs showed a much lower level of gene expression than UP7CNs. Possibly, fewer positive charges of UP25CNs resulted in weaker electrostatic attractions between pDNA and UA-PAEP25 after UP25CNs escaped from endo-

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Figure 5. Transfection efficiency of Hela cells mediated by PACNs at various polymer/DNA ratios (N/P) in serum-free DMEM, PEICNs at the N/P ratio of 20 as control group (A). The transfection efficiency of PACNs prepared in water for Hela and COS 7 cells (B). Transfection efficiency of PACNs, UPCNs, and PEICNs prepared in water or acetate pH 5.7 at N/P ratio of 35 for Hela (C) and COS 7 cells (D) (n ) 3, error bars represent standard deviation). The transfection efficiency was expressed as total fluorescence intensity of 1 × 104 cells. *p < 0.05 compared with the transfection of complex nanoparticles prepared in water.

Figure 6. Effect of proton pump inhibitor bafilomycin A1 on transfection mediated by polymer/DNA complex nanoparticles at the polymer/DNA ratio (N/P) of 35. COS 7 cells and Hela cells were transfected in the presence of 200 nM or without Bafilomycin A1, respectively. The transfection efficiency was expressed as total fluorescence intensity of 1 × 104 cells (n ) 3). **p < 0.01 or *p < 0.05 compared with the transfection of complex nanoparticles in the presence of bafilomycin A1.

some into cytosol with higher pH, thus inducing DNA to dissociate with complex nanoparticles and to be hydrolyzed by lysosomal enzyme. Therefore, the poor transfection efficiency of UP25CNs might be attributed to instability resulting from low zeta potential rather than buffering capacity. However, the absence of endosomal escape could be one of reasons accounting

for unsatisfactory transfection activity of UP25CNs compared with UP7CNs as well. As for PACNs with the highest zeta potential, inferior buffering capacity might be one of the factors leading to a lower level of gene transfection than UP7CNs. The acetate solution (20 mM, pH 5.7) improved the transfection activity of UPCNs (especially UP25CNs) in Hela cells

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Figure 7. Cytotoxicity of PACNs and UPCNs at polymer/DNA ratio (N/P) on COS 7 cells and HeLa cells measured by MTT assay. PEICNs as control (n ) 3, error bars represent standard deviation). **p < 0.01 compared with the cytotoxicity of PEICNs.

(Figure 9) and COS 7 cells, while this effect was not observed in the case of PACNs. The results suggested that the increased transfection efficiency of UPCNs in 20 mM acetate (pH5.7) could be due to increased positive charges and the enhancement of the protonation from imidazole moieties, especially for UP25CNs with higher UA denstity. However, for PACNs, a permanent positive charge from basic amino groups induced the little alteration of the stability in cytosol as well as the transfection activity without regard to pH of preparation medium. Bafilomycin A1, a proton pump inhibitor, is used to testify whether the buffering-based endosomolytic activity plays

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an important role on the transfection efficiency. Bafilomycin A1 is an antibiotic that selectively inhibits vacuolar type of H+ATPases (25). Figure 6 showed that the involvement of Bafilomycin A1 resulted in a significant decrease in the gene expression levels of PACNs and UPCNs in Hela and COS 7 cell lines, which suggested that the transfection efficiencies of all the above complex nanoparticles were dependent on the endosomal acidification process. Because of this, it is very meaningful to conjugate the UA moiety with PAEP to increase the buffering capacity, which benefits the endosomal escape of complex nanoparticles to improve the transfection efficiency. Even if, in the presence of a proton pump inhibitor, the transfection activity of UP7CNs was still higher than that of other complex nanoparticles, which also meant that the marginally increased buffering capacity of UA-PAEP7 was not enough to explain its higher transfection efficiency. Cytotoxicity of Polymer/DNA Complex Nanoparticles. The cytotoxicity of complex nanoparticles at polymer/DNA ratio (N/ P) of 35 in COS 7 and Hela cells was shown in Figure 7. In COS 7 cells, the cell viability of PACNs was 69%, which was slightly higher than that of PEICNs. When PAEP was modified by UA, the cell viability of complexed nanoparticles increased from 81% to 88% with the substitute degree of UA from 7% to 25%. The descending cytotoxicity of polymer modified by UA could be due to the decreased positive charges. The similar results of cytotoxicity also occurred in Hela cells. So, it was obvious that the cytotoxicity of UPCNs was much lower than that of PEICNs in both cell lines.

Figure 8. Cellular uptake of nanoparticles prepared in water or acetate (pH 5.7) on COS 7 cells (A) and Hela cells (B). The cells were incubated for 1 h with YOYO-1-labeled nanoparticles. After a subsequent washing and trypsinization, the mean fluorescence intensity (MFI) was analyzed with a flow cytoflurometer. MFI of PEICNs was normalized to 100. Nanoparticles were prepared at the N/P ratio of 35. Cellular uptake of PACNs, UP7CNs, or UP25CNs by Hela cells after incubation with various inhibitors (C). Percentages of cellular uptake expressed as % of control were means ( SD from three independent experiments. *Values significantly different from control (p < 0.05).

Figure 9. Images of Hela cells transfected with PACNs, UP7CNs, UP25CNs, and PEICNs prepared either in 20 mM acetate (pH 5.7) (A,B,C,D) or in distilled water (E,F,G,H) observed under fluorescent microscope (20× magnification) at the N/P ratio of 35.

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Cell Uptake of Polymer/DNA Complex Nanoparticles. Uptake of polymer/DNA complex nanoparticles through endocytosis into the cells is the first step during intracellular gene delivery. It is essential to investigate the cellular uptake of complex nanoparticles for more insight into the diversity of transfection activity among three complex nanoparticles. The amount of pDNA internalized by Hela or COS 7 cells was estimated after quenching the fluorescence of the complex nanoparticles adsorbed on the cell surface by adding trypan blue to the cell suspension in PBS. The PACNs, UPCNs, and PEICNs were prepared at the polymer/DNA ratio (N/P) of 35. As shown in Figure 8A, the DNA uptake efficiency mediated by UP7CNs prepared in either distilled water or 20 mM acetate (pH 5.7) was much higher than that of other complex nanoparticles under same condition in COS 7 cell lines. So, the higher cell uptake level of UP7CNs could be one of the reasons for their superior transfection activity. Although the uptake level of PEICNs was lower than UP7CNs, the strong buffering capacity of PEI 25K, which would benefit intracellular trafficking of nanoparticles, could result in its higher transfection efficiency than UP7CNs. Although UPCNs prepared in 20 mM acetate (pH 5.7) showed enhancement of transfection activities than that prepared in water, no significant difference in terms of DNA uptake of complex nanoparticles between different preparation systems was observed. The results indicated that a weak acid preparation system could improve the compaction of UPCNs rather than their cellular uptake. In Hela cell lines, the uptake of complex nanoparticles prepared in 20 mM acetate (pH 5.7) was similar to that in COS 7 cells, while the DNA uptake level mediated by UP25CNs prepared in distilled water surprisingly was the highest among the three complex nanoparticles. Current evidence suggests that the positively charged polymer/DNA complex nanoparticles are taken up by means of endocytosis (26, 27). Since different endocytic pathways exist, it is important to have a more detailed knowledge of the cellular uptake and routing mechanism of polymer/DNA complex nanoparticles. To know whether the type of endocytosis would bring about the above phenomenon of UP25CNs, quantitative measurement of the uptake of complex nanoparticles in the presence of endocytosis inhibitors was investigated in Hela cell lines. In our experiments, disruption of macropinocytosis by means of cytochalasin D decreased all of PACN, UP7CN, and UP25CN uptake to less than 30%, as a percentage of control (i.e., without preincubation with inhibitor) (Figure 8C). Inhibition of caveolaemediated uptake by genistein, an inhibitor of tyrosine kinases involved in caveolae-mediated endocytosis, inhibited uptake of UP7CNs and UP25CNs by 67% and 70%, respectively, while the internalization of PACNs was significantly reduced by almost 50%. Inhibition of clathrin-mediated uptake by chlorpromazine and microtubule-mediated by nystatin did not significantly reduce the uptake of PACNs, UP7CNs, and UP25CNs. These results demonstrated that macropinocytosis dominated the uptake routes of three complex nanoparticles. Caveolae-mediated endocytosis was also involved in the uptake of PACNs, while clathrin-mediated and microtubulemediated pathways were not involved in the uptake of three complex nanoparticles. There was no significant difference in endocytic pathway between UP7CNs and UP25CNs. It seemed likely that the improvement of transfection mediated by UP7CNs was related to the following parameters including ζ-poptential, pH buffering capacity, and cellular DNA

uptake. However, the intracellular trafficking and nuclear uptake are two crucial processes involved in enhanced gene expression of UP7CNs.

CONCLUSION A biodegradable cationic polyphosphazene with (2-(2aminoethoxy)ethoxy) as side chains bearing primary amino groups was synthesized and first used for gene delivery. PACNs could mediate efficient transfection in complete medium at the polymer/DNA ratio (N/P) of 35. UP7CNs could enhance the transfection efficiency compared with PACNs and decrease cytotoxicity compared with PEICNs in HeLa and COS 7 cell lines. The transfection experiment using a proton pump inhibitor suggested that the gene expression of PACNs, UPCNs were dependent on the endosomal acidification process. The acetate solution (20 mM, pH 5.7) could further improve the transfection activity of UPCNs, which was almost comparable to PEICNs in HeLa and COS 7 cell lines. So, UP7CNs could be a more potential carrier for gene delivery. Further studies would focus on evaluating in vivo transfection efficiency of UP7CNs.

ACKNOWLEDGMENT The National Basic Research Program of China (nos 2007CB935800 and 2009CB930300), the National Natural Science Foundation of China (30873169), the Important Direction Program of CAS (KJCX2.YW.M02, KSCX1-YW-R-21, KSCX2-YW-R-09), and Shanghai Nanomedicine Program (0852 nm05700) are gratefully acknowledged for financial support.

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