Polyamidoamine Dendrimers with a Modified Pentaerythritol Core

Feb 12, 2009 - Polyamidoamine (PAMAM) dendrimers represent one of the most efficient polymeric gene carriers. This study describes a new family of ...
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Biomacromolecules 2009, 10, 617–622

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Polyamidoamine Dendrimers with a Modified Pentaerythritol Core Having High Efficiency and Low Cytotoxicity as Gene Carriers Yanming Wang,†,‡ Weiling Kong,† Yu Song,† Yajun Duan,† Lianyong Wang,† Gustav Steinhoff,§ Deling Kong,*,† and Yaoting Yu† Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Science, Nankai University, Tianjin 300071 China, College of Pharmacy, Nankai University, Tianjin 300071, China, and Department of Cardiac Surgery, University of Rostock, Rostock, Germany Received November 18, 2008; Revised Manuscript Received January 21, 2009

Polyamidoamine (PAMAM) dendrimers represent one of the most efficient polymeric gene carriers. This study describes a new family of PAMAM dendrimers that can be synthesized using a Pentaerythritol derivative (PD) as a core that possesses 12 branches. This new approach in the synthesis of divergent dendrimers provided a rapid increase in the number of branches, which made it easier to obtain dendrimers with high generation and large enough molecular size. The PD dendrimers of generations 3-5 synthesized in this study could efficiently condense DNA into nanoscale complexes with slightly positive charges. Their transfection efficiency was evaluated in different cell lines. These PD dendrimers were found to show higher transfection efficiency, but much lower cytotoxicity, than the commercial nonviral gene carriers polyethyleneimine (PEI), polylysine (PLL), and PAMAM dendrimers with an ethylenediamine core (generations 5 and 7). The results indicate that, with high transfection efficiency and low cytotoxicity, the PD dendrimers hold promise as novel nonviral gene carriers.

Nonviral delivery systems for gene therapy have been increasingly proposed as safer alternatives to viral vectors.1,2 These vectors induce no or minimal host immune responses, allowing repeat administrations. They can be modified for targeting purposes and easily produced in large quantities. Among the nonviral vectors, several synthetic and natural cationic polymers, such as cationic lipid,3 PLL,4 PEI,5 chitosan,6 and so on, have been intensively studied. Although some cationic polymers showed exciting efficiency in cell culture, their relatively low transfection efficiency in vivo and inherent cytotoxicity limited their use as gene carriers for clinical treatment.7,8 Nevertheless, cationic polymers are still attractive to many scientists because of their well-defined structure and easy control of surface functionality for certain biomedical applications.9 PAMAM dendrimers are typically a class of well-defined, highly branched, and monodispersed polymers that carry amino groups on their surface. They have exhibited high transfection efficiency in a variety of suspensions and adherent cultured mammalian cells.10-12 Their precise and controllable structures, with relatively low toxicity, have fueled many studies on new molecular design and intensive gene delivery tests both in vitro and in vivo.13,14 The primary amines located on the surface of PAMAM provide a structural basis to conjugate suitable ligands, such as transferrin15 and folic acid,16 for tumor-targeted gene delivery. A recent study showed that conjugation of the PAMAM dendrimer to the surface of poly(lactic-co-glycolic acid) (PLGA) microparticles eliminated the generation-depend-

ent cytotoxicity in HEK293 and COS-7 cell lines and significantly increased the transfection efficiency in comparison with unmodified PLGA microparticles.17 Studies have also shown that the density of amino groups on the dendrimer surface, the size, and generation of the dendrimers are important parameters affecting the gene delivery efficiency.18 On the other hand, these parameters are dependent on the core from which the dendrimers are derived. PAMAM dendrimers derived from different cores, although sharing similar chemical structures and being of the same generation, have different charge densities on the surface and exhibit different gene delivery capacities.19 Baranov’s group reported that particle size is one of the most critical parameters affecting the transport transfection behaviors of the dendrimer/DNA complexes in vivo and in vitro.20 However, the synthetic efforts to obtain dendrimers of high generations are often laborious (12-18 steps).21,22 A previous study has focused on developing simple synthetic routes to dendritic polyamines with defined structures.11 In this paper, we describe a new approach for the synthesis of PAMAM dendrimers. We used a Pentaerythritol derivative (PD) as the core, which bears twelve amino groups stretching outward, thus enabling us to construct a dendrimer with a rapid increase in the number of branches. The PD dendrimers were synthesized and characterized up to generation 5 (G5). The physical and chemical properties of three PD dendrimers (G3 to G5) in terms of DNA complexation, particulate properties, surface charge, and transfection efficiency were investigated and compared with commercial gene vectors, such as PEI, PLL, and G5 and G7 PAMAM dendrimers with an ethylenediamine core.

* To whom correspondence should be addressed. Fax: 0086-22-23498775. E-mail: [email protected]. † College of Life Science, Nankai University. ‡ College of Pharmacy, Nankai University. § University of Rostock.

Materials. Pentaerythritol, tris(hydroxymethy1) aminomethane (Tris), Poly-L-lysine (PLL, MW 27 kDa), polyethyleneimine (branched PEI;

Introduction

Experimental Section

10.1021/bm801333s CCC: $40.75  2009 American Chemical Society Published on Web 02/12/2009

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MW 25 kDa, and G5 and G7 PAMAM dendrimers with an ethylenediamine core (EDA dendrimers) were purchased from Sigma (St. Louis, MO). 3-{4,5-Dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, and ampicillin were purchased from Gibco BRL (Grand Island, NY). DNase I was obtained from Takara (Dalian, China). The plasmid pEGFP-C1 was kindly provided by Dr. Cunxian Song (Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, Tianjin, China). QIAGEN Maxiprep plasmid purification kit was purchased from Qiagen (Hilden, Germany). Methyl acrylate was redistilled prior to use. Methanol was distilled from magnesium and ethylenediamine was distilled from calcium hydride. All other organic solvents were of analytical reagent grade. Synthesis of Dendrimers. Generation 0.5. A 12-directional pentaerythritol-derived core molecule was first synthesized following Bruson23 and Newkome’s24 procedures. The product (1.40 g, 0.62 mmol) was then dissolved in 70 mL of methanol and was added dropwise to 3 mL of methyl acrylate (2.87 g, 33.35 mmol) under N2. Sodium methoxide (0.016 g, 0.3 mmol) was added as a catalyst and the mixture was stirred at room temperature for 48 h. The solvent and excess methyl acrylate were removed in vacuum to give a yellow oil (3.2 g, 89%) that was denoted as G0.5 PD dendrimer. Generation 1. To a solution of G0.5 PD dendrimers (2.4 g, 0.56 mmol) in 50 mL of methanol was added dropwise 15 mL of ethylenediamine (13.47 g, 0.22 mol), which was cooled in an ice-water bath. The mixture was then stirred at room temperature for 72 h under N2. Ethylenediamine was removed by evaporation. Further purification was performed by coevaporation after dissolving the residues in 20 mL of methanol/diethyl ether (v/v 1:1). The coevaporation process was repeated 2-3 times. A yellow oil was obtained (3.1 g, 96%), and was denoted as G1 PD dendrimer. Generations 2-5. PD dendrimers of G2-G5 were synthesized by repeating the above two steps as shown in Scheme 1. Dendrimers Characterization. 1H NMR was performed on a nuclear magnetic resonance spectrometer (Varian Mercury VX 400 MHz) using chloroform-d or dimethyl sulfoxide-d6 as a solvent for half or full generation PD dendrimers, respectively. Samples were pressed into KBr pellets, and Fourier transform infrared (FT-IR) spectra were obtained using a Bio-Rad FTS600 infrared spectrometer. Number- and weight-average molecular weights (Mn and Mw, respectively) were determined by gel permeation chromatography (GPC) at 30 °C using a Waters 410 HPLC module which was equipped with a Shodex KB803 column. Sodium nitrate buffer (0.15M, pH 7.4) was used as the mobile phase at a rate of 0.3 mL/min. Near-monodisperse polyvinyl alcohol standards (Sigma) were employed for calibration. 1 H NMR (400 MHz, DMSO-d6, δ (ppm)): for G3 PD dendrimers, 1.3-1.8 and 2.2-2.4 (m, other -CH2-), 2.6-2.8 (t, -CONHCH2-), 3.2-3.4 (m, OCH2), 7.3-7.6 (m, -CONHCH2-); for G4 PD dendrimers, 1.4-1.7 and 2.3-2.5 (m, other -CH2-), 2.7-2.9 (t, -CONHCH2-), 3.3-3.4 (m, OCH2), 7.5-7.8 (m, -CONHCH2-); for G5 PD dendrimers, 1.6-1.9 and 2.3-2.5 (m, other -CH2-), 2.7-2.9 (m, CONHCH2), 3.2-3.5 (m, OCH2), 7.8-8.1 (m, -CONHCH2-) (see Supporting Information for the spectra). 13 C NMR in D2O was used to determine the efficacy of branching. This is further described in the Supporting Information section. Acid-Base Titration. The buffer capacity of G3-G5 PD dendrimers in the pH range of 4-10 was determined by acid-base titration. Dendrimers were dissolved in saline (0.9% NaCl solution) to a concentration of 8 mM (primary amino groups), and the solutions were then titrated with a 0.1 N HCl solution and monitored with a pH meter. The pKa values of G3-G5 dendrimers were obtained from the pH titration profiles.19 Cell Culture. Mouse embryonic Fibroblast cells (NIH 3T3), monkey kidney Fibroblast cells (COS-7), human breast cancer cells (MCF-7), and human hepatocellular liver carcinoma cells (HepG2) were cultured

Wang et al. in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, and 4 mM L-glutamine at 37 °C in a humidified 5% CO2. Preparation of Plasmid DNA. The pEGFP-C1 plasmid was amplified by transformation of E. coli, growing of the bacteria and isolation of the plasmid with the endotoxin-free QIAGEN Maxiprep kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s protocol. The purity and concentration of the plasmid DNA were determined by ultraviolet (UV) absorbance at 260 and 280 nm. Gel Retardation Assay. The formation of dendrimer/DNA complexes was examined by their electrophoretic mobility on agarose gel. Plasmid DNA (1 µg) was mixed with the dendrimers in 8 µL of phosphate buffered saline (PBS) at various concentrations corresponding to molar ratios between amino groups and phosphate groups (N/P ratios) ranging from 0 to 15. The mixture was incubated for 30 min at room temperature and then analyzed on 0.8% (w/v) agarose gel stained with ethidium bromide (EB, 0.5 µg/mL) for 40 min at 80 V. The gel was then examined with a UV illuminator for the mobility of DNA bands. Protection of DNA from DNase I Digestion. Each dendrimer/DNA complex at N/P ratio of 5:1 was equally divided into triplicates. One was used as a control and the other two were incubated with DNase I (1 unit per µg DNA) at 37 °C for 2 h. Then, 3 µL of sodium dodecyl sulfate (10% SDS, w/v) was added to one of the DNase I-treated samples to recover the DNA. Naked DNA samples, with and without the DNase I treatment, were used as controls. Agarose gel (0.8%, w/v) electrophoresis was performed to evaluate the integrity of DNA in the complexes. Particle Size and Zeta Potential. The particle size and zeta potential of the dendrimer/DNA complexes were measured using a BI-90 plus Zetasizer. Dendrimer/DNA complexes were prepared by mixing 16 µg of plasmid DNA with dendrimers in 3 mL of PBS at different N/P ratios. The mixture was incubated for 30 min at room temperature. The particle size was measured at a 90° scattering angle, and the mean hydrodynamic diameter was determined by cumulative analysis. The zeta potential measurements were performed automatically using an aqueous dip cell. Cytotoxicity Assay. The cytotoxicity of the PD dendrimers in comparison with PEI, PLL, and G5, G7 EDA dendrimers was evaluated by MTT assay.25 NIH 3T3 cells were seeded in 96-well plate at a density of 104 cells per well. After 24 h, the medium was removed and replaced with 100 µL of DMEM containing different concentrations of PD, PEI, PLL, or EDA dendrimers. Each concentration was replicated in six wells. The cells were allowed to grow for an additional 24 h. Then 25 µL of MTT (5 mg/mL in PBS) was added to each well. After 2 h at 37 °C, 100 µL of extraction buffer (20% w/v SDS in 1:1 DMF/water, pH 4.7) was added. After an overnight incubation at 37 °C, the optical density of each well was measured at 550 nm. The viability of cells was expressed as a percentage relative to the control wells. In Vitro Transfection and Expression Assay. The transfection efficiency of G3-G5 PD dendrimers was evaluated using plasmid pEGFP-C1 that expresses green fluorescence protein (GFP). On a 24-well plate, 80000 cells (MCF-7 or HepG2) or 60000 cells (NIH 3T3 or COS-7) per well were seeded 24 h before the transfection experiment. The cells were washed with PBS and replaced with 250 µL of serum-free medium. Plasmid DNA (2 µg per well) was mixed with dendrimer solutions in 250 µL of serum-free medium at different N/P ratios from 1:1 to 20:1. The mixture was incubated for at least 15 min at room temperature to allow the formation of dendrimer/DNA complexes. The complexes were then added into the well and incubated with the cells for 4 h at 37 °C. The medium was removed and replaced with fresh complete medium. After 48 h at 37 °C the cells were analyzed for protein expression. GFP expression was analyzed by flow cytometry (FACS calibur, Becton Dickinson, UK). For PEI, PLL, and G5 and G7 EDA dendrimers,

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Scheme 1. Synthetic Scheme of G1-G5 PD Dendrimers

gene delivery was performed under the optimal conditions described by the manufacturers. Additionally, to investigate the influence of serum on transfection, COS-7 and MCF-7 cells were transfected in

the medium containing various amounts of serum. Each transfection experiment was performed in duplicates and repeated at least twice. Data are reported as mean ( standard deviation.

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Table 1. Molecular Weight of G3-G5 PD Dendrimers generation

theoretical molecular weight

Mw

Mn

PDI

3 4 5

10468 21356 43244

12439 24528 52626

10912 21706 46163

1.14 1.13 1.16

Statistical Analysis. Paired-samples t test was used in this study. P < 0.05 was considered significant.

Results and Discussion Synthesis and Characterization of Dendrimers. The syntheses of G3-G5 PD dendrimers are shown in Scheme 1. First, a pentaerythritol-derived core molecule, which initiated a series of 12-directional-growth dendrimers, was synthesized according to Bruson and Newkome’s method.23,24 The molecule was then reacted with methyl acrylate via Michael addition to yield G0.5 PD dendrimers. G1.0 PD dendrimers were prepared by amidation of G0.5 PD dendrimers with a large excess of ethylenediamine to prevent dendrimer bridging. Repeating Michael addition and amidation resulted in dendrimers of higher generations. The structures of G1-G5 PD dendrimers were confirmed by FT-IR and 1H NMR analyses. In the case of the half-generations (G0.5, G1.5, G2.5, G3.5, and G4.5), the most important IR peaks for structure identification are the ester carbonyl stretching at 1700-1760 cm-1 and the ester C-O stretching peak appearing at around 1260 cm-1. For the full generations, methyl ester groups were converted to amide groups and the corresponding carbonyl peak shifted to 1600-1660 cm-1. The 1H NMR data that was summarized in the synthesis section correlated well with the FT-IR and further confirmed the structure of the PD dendrimers. For example, the ester methyl proton peak at δ 3.8 (data not shown) in the NMR spectra of half-generations was absent in the spectra of the full generations. The molecular weights of G3-G5 PD dendrimers were characterized by GPC, and the results are shown in Table 1. Narrow distributions (PDI ) 1.13-1.16) were observed and suggested that the molecular weights of our synthetic PD dendrimers were close to the theoretical values (see the Supporting Information for the GPC traces). These molecular weights, along with 1H NMR and 13C NMR data, indicate that our PD dendrimers are very close to monodisperse dendrimers. The degree of branching is higher than 95% according to the 13 C NMR data. Generally, perfect monodisperse dendrimers can be easily obtained for low generations, up to G-3. Higher generations may exhibit deviation from absolute monodispersity due to minor structural defects. In this study, we used the derivative of pentaerythritol as a core that possesses 12 branches and is relatively structure-flexible. The growth of dendrimers of generation up to G5 may not lead to detectable structural defects in our case compared to dendrimers grown from an inositol-derived core, which is a more rigid molecule and may be sterically hindered for synthesis of high-generation dendrimers. By using a derivative of pentaerythritol as the core, our synthesis route led to a rapid increase in the number of surface amino groups, reaching 384 at G5, while it is only 128 for ethylenediamine core boronated PAMAM. Buffer Capacity of G3-G5 PD Dendrimers. To investigate the buffer capacity of G3-G5 PD dendrimers, acid-base titration was performed and the pKa values were measured. Figure 1 demonstrates that relatively high buffer capacities of G3-G5 PD dendrimers occurred at two pH ranges. One was from 8.0 to 10.0, corresponding to a pKa1 value of ∼9, and the

Figure 1. Titration curves of G3-G5 PD dendrimers. Dendrimer solutions at 8 mM of amino group concentration were titrated with 0.1 N HCl. The constant d[H]/dpH, which is defined as the buffer capacity, is plotted against the pH value of the solution.

Figure 2. (A) Gel electrophoresis of PD dendrimer/DNA complexes. Lane 1: naked DNA; lanes 2-7: G3 PD dendrimer/DNA complexes; lanes 8-13: G4 PD dendrimer/DNA complexes; lanes 14-19: G5 PD dendrimer/DNA complexes. For each PD dendrimer, from left to right, N/P ratios are 0.5:1, 1:1, 2:1, 5:1, 10:1, and 15:1, respectively. (B) DNase I digestion of dendrimer/DNA complexes. Lane 1: DNA ladder; lane 2: naked DNA; lane 3: naked DNA treated with DNase I; lanes 4-6: G3-G5 PD dendrimer/DNA complexes at N/P ratio 5 without DNase I and SDS treatment; lanes 7-9: G3-G5 PD dendrimer/DNA complexes at N/P ratio 5 treated with DNase I; lanes 10-12: G3-G5 PD dendrimer/DNA complexes at N/P ratio 5 first treated with DNase I and then followed by SDS treatment. Dendrimer/ DNA complexes were incubated with DNase I at 37 °C for 2 h, then were loaded on 0.8% (w/v) agarose gel.

other one was from 5.0 to 7.0, corresponding to a pKa2 value of ∼6. It indicates that G3-G5 PD dendrimers are able to buffer the pH change in the weak acidic endosomal environment.26 This should allow the PD dendrimers to buffer the endosome lumen and escape the endolysosomal compartment after cellular internalization. Our results are consistent with a previous study,19 which reported a similar buffer capacity in two pH ranges, 8.7∼9.4 and 5.5∼6.4. Stability of Dendrimer/DNA Complexes. The formation of dendrimer/DNA complexes was examined using agarose electrophoresis. Figure 2A shows the gel retardation results of dendrimers/DNA complexes at different N/P ratios. Complete retardation of DNA was achieved at N/P ratios of 1 or 2 for all three dendrimers. This suggests that these PD dendrimers have similar capacity to form complexes with DNA. However, differences in the EB fluorescence were found among them. The G3 dendrimer showed EB fluorescence in samples of N/P ratio 0.5:1, 1:1, 2:1, and 5:1, while no fluorescence was observed in samples of high N/P ratio (10:1, 15:1). In contrast, for G5 dendrimer, fluorescence quenching was observed when the N/P ratio was as low as 2:1. This is because the G5 dendrimer has the highest density of positive charge and exhibits the strongest interaction with DNA. As a result, such a strong interaction

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Figure 3. Characterization of PD dendrimer/DNA complexes. (A) The particle size of G3-G5 PD dendrimer/DNA complexes. (B) Zeta potential of G3-G5 PD dendrimer/DNA complexes. Each result represents the mean value of three runs.

Figure 4. Cytotoxicity of G3-G5 PD dendrimers tested with NIH 3T3 cells. Relative cell viability was measured by MTT. Data represent mean ( standard deviation (n ) 6).

may prevent the binding between DNA and EB and lead to fluorescence quenching. The DNase I digestion assay indicated that all three PD dendrimers could form complexes with DNA and protect the DNA from DNase I digestion. Free DNA was completely digested by DNase I and no DNA band can be seen (Figure 2B, Lane 3). In the absence of DNase I and SDS, dendrimer/DNA complexes were retarded in the gel and the fluorescence was quenched (lanes 4-6, same results as in Figure 2A). Treatment with DNase I did not show any DNA band and EB fluorescence (lanes 7-9). However, with SDS added to the solution after DNase I digestion, DNA was released from the complexes and remained unchanged (lanes 10-12). These data clearly demonstrate that our dendimers have the capacity to form complexes with DNA and protect DNA from enzyme digestion. Particle Size and Zeta Potential of Dendrimer/DNA Complexes. PD dendrimers are able to form complexes with plasmid DNA via electrostatic interaction. However, the complex-

ation appears to be greatly dependent on dendrimer generation. As shown in Figure 3A, G4 and G5 PD dendrimers could compact DNA into nanoparticles of 200-300 nm in diameter while the particle size of G3/DNA complexes was bigger than 400 nm. At the same N/P ratio, the particle size decreased with the increase of generation number. The results indicate that an increase in density of surface charge is favorable to the efficient compaction of dendrimers with DNA. To estimate the surface charge of dendrimer/DNA complexes, zeta potential was measured in low ionic solution and the results are shown in Figure 3B. The zeta potential was above 20 mV for G4 and G5 PD dendrimers at N/P ratios from 5 to 15, while it is lower (less than 10 mV) for G3 PD dendrimer. The N/P ratio affected the biophysical properties of the dendrimer/DNA complexes. The net surface charge of the particles increased from negative to positive as the N/P ratio increased. It is well-known that positive zeta potential is critical for polymer-based gene delivery systems since the cationic microparticles demonstrate an enhanced interaction with the negatively charged cellular membranes that is most likely responsible for the enhanced cell uptake.17 Cytotoxicity of G3-G5 PD Dendrimers. The cytotoxicity of PD dendrimers was evaluated in cultured NIH 3T3 cells by MTT assay. As shown in Figure 4, the cytotoxicity of the PD dendrimers was independent of dendrimer generation and was much lower than that of the reference cationic polymers PEI and PLL. The cytotoxicity of the PD dendrimers was also lower than that of two other commercial dendrimers, namely, G5 and G7 EDA dendrimers. Cytotoxicity has been a problem for PEI in spite of its excellent transfection efficiency. The need of safe gene carriers has fueled a lot of studies on new molecular designs and poly(ethylene glycol) (PEG)27 or cyclodextrin28 modification of PEI. Our PD dendrimers showed reduced cytotoxicity compared with commercial EDA dendrimers. Although what made the difference is still under

Figure 5. Transfection efficiency of PD dendrimers. (A) The effect of N/P ratio on the transfection efficiency of G3-G5 PD dendrimers was analyzed in COS-7 cells. DNA (2 µg) was applied to each well on a 24-well plate. The N/P ratio for PLL and PEI was 1:1 as suggested by the manufacturer (*P < 0.05, G5 PD dendrimer vs PEI and PLL). (B) Transfection efficiency of G5 PD dendrimer was evaluated in different cell lines. Cells were incubated with complexes containing 2 µg of pEGFP-C1 plasmid DNA in the absence of serum. The N/P ratio for PLL and PEI was 1:1, but 10:1 for G5, G7 EDA dendrimers and G5 PD dendrimer. Transfection efficiency was examined by flow cytometry assay at 48 h. Data represent mean ( standard deviation (n ) 3; *P < 0.05, G5 PD dendrimer vs PEI, PLL and EDA dendrimers in different cell lines).

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Conclusions This study described a synthetic approach by which PAMAM dendrimers can be prepared using a derivative of pentaerythritol as the core. This new approach provided a rapid increase of the number of branches, which made it easier to obtain dendrimers with high generation and large enough molecular size for efficient transfection. The PD dendrimers synthesized in this study exhibited high transfection efficiency with negligible cytotoxicity. This suggests that the PD dendrimers are very valuable for further investigation as promising nonviral gene carriers. Figure 6. Effect of FBS on the transfection efficiency of G5 PD dendrimers. COS-7 and MCF-7 cells were transfected with G5 PD dendrimer/DNA complexes at N/P ratio of 10. Data represent mean ( standard deviation (n ) 3).

investigation, the unique core structure and the flexibility of the PD dendrimers may play some roles. In Vitro Transfection. Transfection efficiency of PD dendrimers was evaluated using different cell lines. As shown in Figure 5A, the transfection efficiency mediated by PD dendrimers was largely dependent upon the generation number in the order of G5 > G4 > G3. The transfection efficiency of G3 PD dendrimer was much lower than that of G4 and G5. It is probably due to its poor capability to condense DNA into nanoparticles. In addition, as shown in Figure 5A, the transfection efficiency of the three dendrimers increased with the increase of N/P ratio. Compared with the two commercial gene carriers PLL and PEI, all the three PD dendrimers showed higher transfection efficiency than PLL. Furthermore, G4 and G5 PD dendrimers showed higher efficiency than PEI, which is recognized as the most efficient nonviral gene carrier up to date. Given the fact that PLL and PEI are different from PAMAM dendrimers in terms of molecular structure, another two commercial PAMAM dendrimers, G5 and G7 EDA dendrimers, were also used as references. G5 PD dendrimer showed higher transfection than G5 and G7 EDA dendrimers (Figure 5B). A previous study has shown that the core is important for a PAMAM dendrimer.19 The number of functional groups on the dendrimer surface, diameter, and molecular conformation are all dependent on its core. In this study, tris modified Pentaerythritol which has 12 terminal amino groups was used as the core for further divergence. Such unique core enabled rapid increase in the number of terminal amino groups. G5 PD dendrimers showed higher transfection efficiency than G7 EDA dendrimer although the later has more amino groups (512) on the surface. It indicates that the different core structure may play an important role in gene transfection. Effect of Serum on Transfection Efficiency. One of the disadvantages of nonviral gene carriers is that serum inhibits gene transfection in vitro and in vivo. The serum is known to affect the stability of polymer/DNA complexes. In the present study, experiments were carried out in medium containing 0, 5, or 10% FBS, respectively. Figure 6 shows that the presence of serum reduced the transfection efficiency of the PD dendrimers. When the concentration of FBS was 10%, the transfection efficiency was only half of that in serum-free medium. Such a decrease in transfection efficiency has been a common problem for nonviral gene carriers. Several studies have shown that modification with PEG can significantly improve the transfection in vivo.29 Further modification of our PD PAMAM dendrimers with PEG or conjugation of other elements could allow circumventing the inhibition by serum, thus improving in vivo gene delivery.

Acknowledgment. This research was supported by Natural Science Foundation of China (No. 50803029, 20774050) and by National Outstanding Youth Fund (No. 30725030). Supporting Information Available. GPC traces of G3-G5 PD dendrimers along with 13C NMR and 1H NMR spectra of G3-G5 PD dendrimers. This material is available free of charge via the Internet at http://pubs.acs.org.

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