Photoluminescent Hyperbranched Poly(amido amine) Containing β

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Photoluminescent Hyperbranched Poly(amido amine) Containing β-Cyclodextrin as a Nonviral Gene Delivery Vector Yan Chen,† Linzhu Zhou,† Yan Pang,† Wei Huang,† Feng Qiu,† Xulin Jiang,‡ Xinyuan Zhu,*,† Deyue Yan,† and Qun Chen*,§ †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China ‡ Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China § Department of Physics & Shanghai Key Laboratory of Functional Magnetic Resonance Imaging, East China Normal University, North Zhongshan Road 3663, Shanghai 200062, People’s Republic of China

bS Supporting Information ABSTRACT: Hyperbranched poly(amido amine)s (HPAAs) containing different amounts of β-cyclodextrin (β-CD) (HPAA-CDs) were synthesized in one-pot by Michael addition copolymerization of N,N0 -methylene bisacrylamide, 1-(2-aminoethyl)piperazine, and mono-6-deoxy-6-ethylenediamino-βCD. In comparison to pure HPAA, the fluorescence intensity of HPAA-CDs was enhanced significantly while the cytotoxicity became lower. Ascribed to plenty of amino groups and strong photoluminescence, HPAA-CDs could be used as nonviral gene delivery vectors, and the corresponding gene transfection was evaluated. The experimental results indicated that HPAA-CDs condensed the plasmid DNA very well. By utilizing the fluorescent properties of HPAA-CDs, the cellular uptake and gene transfection processes were tracked by flow cytometry and confocal laser scanning microscopy without any fluorescent labeling. The transfection efficiencies of HPAA-CDs were similar to that of pure HPAA. In addition, the inner cavities of β-CDs in HPAA-CDs could be used to encapsulate drugs through host
guest interaction. Therefore, the HPAA-CDs may have potential application in the combination of gene therapy and chemotherapy.

’ INTRODUCTION Nonviral gene delivery vectors especially cationic polymers are receiving more and more attention because of their easy manufacture, high compound stability, and low immunogenicity over viral vectors.1
8 To achieve a safe and efficient DNA delivery, the exploration of polyplex formation between the polycations and the plasmid DNA (pDNA) and subsequent cellular uptake and trafficking of genetic materials in the cells is indispensable. By attaching the fluorescent probes onto polycations, the gene delivery process could be tracked. 9
12 However, the fluorescent labeling usually requires multiple complicated reactions. Even worse, the introduction of fluorescent probes might change the physical/chemical properties of gene vectors, resulting in a negative effect on the biocompatibility as well as on the transfection efficiency. Therefore, the preparation and application of photoluminescent polycationic vectors have become very attractive in the field of gene delivery. In recent years, some polycations including poly(amido amine) and polyethylenimine were found to be photoluminescent polymers which could be used for fluorescence imaging.13
20 However, comparing fluorescent materials with traditional fluorophores, r 2011 American Chemical Society

the photoluminescence intensity of these polycations is still very weak.20
22 We have noted that the photoluminescent property of polymers is closely related to their molecular conformations. By enhancement of the chain rigidity, the photoluminescence intensity of polymers can be improved.19 For the traditional linear polycations, only limited end-groups are available for further modification. In contrast to the linear polymers, the highly branched polycations have a great number of various amine groups, which favors the following functionalization. At the same time, the highly branched polycations exhibit low cytotoxicity and high transfection efficiency because of their compact and globular structures.23
27 In the present work, the biodegradable β-cyclodextrin (β-CD) with low cytotoxicity was incorporated into hyperbranched poly(amido amine) (HPAA) by a simple and efficient one-step Michael addition copolymerization. The introduction of big β-CD molecules enhanced the molecular rigidity of HPAA greatly, resulting in a dramatic improvement of Received: January 7, 2011 Revised: April 28, 2011 Published: May 02, 2011 1162

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’ MATERIALS AND METHODS Materials. 1-(2-Aminoethyl)piperazine (AP, 99%), poly(ethylenimine) (PEI, water free, Mw = 25 kDa, Mn = 10 kDa) and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Aldrich. N,N0 -Methylenebis(acrylamide) (MBA, 96%), p-toluenesulfonyl chloride, 1,2ethylenediamine (EDA), acetone, dimethyl sulfoxide (DMSO), sodium hydroxide (98%) were supplied by Shanghai Sinopharm Chemical Reagent Co., Ltd. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), phosphate buffered solution (PBS), penicillin, and streptomycin were purchased from PAA Laboratories GmbH. All these chemicals were used as received without any further purification. Clear polystyrene tissue culture treated 6-well and 96-well plates were purchased from Corning Costar. β-CD was obtained from Shanghai Sinopharm Chemical Reagent Co., Ltd., and purified once by recrystallization from deionized water before use. Mono-6-deoxy-6-ethylenediamino-β-CD (EDA-β-CD) used here was synthesized according to the method reported in our previous work.28 Synthesis of HPAA and HPAA-CDs. Both HPAA and its β-CD derivatives (HPAA-CDs) were synthesized via the Michael addition polymerization in nitrogen atmosphere.28 A typical procedure is as follows: MBA (3.0830 g, 20 mmol) was dissolved in 20 mL of ultrapure water under stirring. Then, AP (2.5190 g, 19.5 mmol) and EDA-β-CD (0.5885 g, 0.5 mmol) were dissolved in 10 mL of ultrapure water and also added into the above solution. The mixture was stirred vigorously at 60 °C in the dark for 60 h. At last, the mixture was precipitated in acetone, dried under vacuum at 50 °C, and a light yellow powder was obtained (4.64 g, yield: 75%). The β-CD content of the polymers (1.3% and 6.1% for the two samples) was determined by quantitative 13 C NMR. Thus, the two samples were named HPAA-CD1.3 and HPAA-CD6.1, respectively. Complexation of HPAA-CDs and Rhodamine B. Rhodamine B (RB) was used as a model drug. Certain amounts of RB were added into HPAA-CD aqueous solutions. After stirring overnight, the RB-loaded polymer solutions were dialyzed against distilled water for 48 h to remove free RB, followed by lyophilization to give the HPAA-CD/RB complexes. Characterization of Polymers. 1H, 13C, 1H
1H COSY, and 13 C
1H HSQC NMR spectra were recorded on a Varian Mercuryplus-400 spectrometer with DMSO-d6 as solvent. The resonance frequencies for 1H and 13C were 400 and 100 MHz, respectively. Quantitative 13C NMR spectra were measured by the method of inverse gated 1H decoupling. The molecular weights and their distributions of the synthesized samples were evaluated by GPC-MALLS (gel permeation chromatography multiangle laser light scattering). The GPC-MALLS system consisted of a Waters 2690D separations module, a Waters 2414 refractive index detector (RI), and a Wyatt DAWN EOS MALLS detector. Two chromatographic columns (Shodex OHpak SB-803 and SB-802.5, Showa Denko, Japan) with a precolumn (Shodex SB-G) were used in series. The sodium acetate (NaAc) solution was prepared by dissolving a calculated amount

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of sodium acetate in reverse osmosis water (0.3 M), and the pH was adjusted to 4.4 with acetic acid. The eluent was filtrated through a 0.22 μm HPLC filter and degassed prior to use by ultrasonic bath. The flow rate of the mobile phase was 0.6 mL/min. The data were processed with Astra software (Wyatt Technology). Fourier transform infrared (FTIR) measurements were performed on a Perkin-Elmer Spectrum 1000 FTIR spectrometer. The UV
vis spectra were recorded on a Perkin-Elmer Lambda 750s UV
vis spectrophotometer. The fluorescence spectra were measured on a QM/TM/RM fluorescence spectrophotometer (Photon Technology International, Inc.). Differential scanning calorimeter (DSC) measurements were conducted on a TA Instruments Q2000 under a nitrogen atmosphere. The heating rate was 20 °C/min. Agarose Gel Electrophoresis Experiments. Each polymer was examined for its ability to bind pDNA through gel electrophoresis experiments. All polymer stock solutions were prepared and mixed with pDNA solution at different N/P ratios (molar ratios of polymer nitrogen to pDNA phosphorus, N/P = 0, 5, 10, 20, 30, 40, 60, and 90) for 30 min at room temperature, and then analyzed on 1% agarose gel containing 0.5 μg/mL ethidiumbromide (EtBr). Then, gel electrophoresis was carried out in Tris-acetate-EDTA (TAE) running buffer with a current of 100 V for 60 min in a Sub-Cell system (Bio-Rad Laboratories, CA). DNA bands were visualized and photographed by a UV transilluminator and BioDoc-It imaging system. Cell Cultures. COS-7 cells (a cell line derived from kidney cells of the African green monkey) were maintained in DMEM (4.5 g/L glucose) supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 units/mL) at 37 °C in a 5% CO2 humidified atmosphere. Confluent cells were subcultured every 3 days using standard procedure. Cell Viability Assay. For MTT assay, COS-7 cells were seeded into 96-well plates at a seeding density of 10 000 cells/well in 200 μL medium. After COS-7 cells were incubated for 24 h at 37 °C and 5% CO2, the adherent cells reached approximately 60
70% confluence. Growth medium in the 96-well plates was then removed carefully by a transfer pipette and replaced with 200 μL medium containing serial dilutions of the polymers. The cells were grown for 24 h. Then, 20 μL of 5 mg/mL MTT assays stock solution in PBS was added to each well. After incubating the cells for 4 h, the medium containing unreacted dye was removed by a transfer pipette carefully. The obtained blue formazan crystals were dissolved in 200 μL/well DMSO and measured spectrophotometrically in a Perkin-Elmer 1420 Multilabel counter at a wavelength of 490 nm. Cell Internalization. The cellular uptake experiments of HPAA-CD/pDNA complexes were characterized by flow cytometry and confocal laser scanning microscopy (CLSM). Flow cytometry was used to provide statistics on the uptake of HPAACD/pDNA into COS-7 cells. COS-7 cells (5.0  105 cells per well) were seeded in a 6-well tissue culture plate in 1 mL complete DMEM and cultured for 24 h until the adherent cells reached approximately 60
70% confluence, followed by removing the culture medium by a transfer pipette and replacing with fresh and prewarmed DMEM in the absence of 10% FBS. Then, the HPAA-CD/pDNA complexes in PBS (corresponding to 4 μg plasmid/well, N/P = 30) were added to different wells and the cells were incubated at 37 °C for predetermined time intervals. After the incubation, samples were prepared for flow cytometry analysis by removing the cell growth medium by a transfer pipette, rinsing with PBS buffer, and treating with trypsin. 1163

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Scheme 1. Synthetic Procedure of HPAA-CDs

Data for 1.0  104 gated events were collected and analysis was performed by means of a BD FACSCalibur flow cytometer and CELLQuest software. For CLSM studies, COS-7 cells were seeded in 6-well plates at 2  105 cells/well in 1 mL complete DMEM and cultured for 24 h, followed by removing culture medium from the 6-well plates and washing with DMEM in the absence of 10% FBS. Then, the HPAA-CD/pDNA complexes (1 mL DMEM medium in the absence of 10% FBS) at the plasmid concentration of 4 μg/mL were added to each well. After incubation at 37 °C for predesignated time, culture medium was removed carefully by a transfer pipette and cells were washed with PBS three times. Then, the cells were fixed with 4% formaldehyde for 30 min at room temperature, and the slides were rinsed with PBS three times. Finally, the cells were stained with 2-(4-amidinophenyl)6-indolecarbamidine dihydrochloride (DAPI) for 10 min and again the slides were rinsed with PBS three times. The slides were mounted and observed by a LSM 510META. In Vitro Transfection Assay. For transfection studies, cells were seeded at a density of 1  105 cells/well in 96-well plates and incubated for 16
24 h until 60
70% confluent at 37 °C and 5% CO2. Immediately prior to transfection, the medium was removed by a transfer pipette. Then, cells were washed and replaced with fresh and prewarmed DMEM in the presence or absence of 10% FBS. The HPAA-CD/pDNA complexes (corresponding to 0.2 μg plasmid/well) were added to each well and the cells were incubated at 37 °C for 4 h. The medium was then replaced with fresh DMEM supplemented with 10% FBS and incubated for additional 48 h. The luciferase assay was carried out according to manufacturer’s protocol (Promega). Relative light units (RLU) were measured with Varioskan Flash (Thermo Scientific, USA) GloMaxTM 96 microplate luminometer (Promega, USA). The obtained RLUs were normalized with respect to protein concentration in the cell extract determined using the BCA protein assay kit (Beyotime, China).

’ RESULTS AND DISCUSSION Synthesis of HPAA-CDs. As mentioned in the introduction part, poly(amido amine) is a kind of cationic polymers with photoluminescence. Unfortunately, its photoluminescence is very weak. To improve its photoluminescence intensity, we introduced big β-CD molecules onto the surface of HPAA to enhance the molecular rigidness. The synthetic procedure of HPAA-CD is shown in Scheme 1. The monomers MBA, AP, and EDA-β-CD with an equal feeding molar ratio of MBA to AP/ EDA-β-CD but different molar ratio of AP to EDA-β-CD (see Table 1) were mixed in water at 60 °C. After reaction for 60 h, HPAAs with different β-CD content (HPAA-CDs) were synthesized through Michael addition copolymerization. The experimental conditions and corresponding characterization data are summarized in Table 1, which confirms the formation of polymerized products. The actual β-CD content was calculated from quantitative 13C NMR spectra. It is found that the β-CD contents of the polymers are 1.3% and 6.1%, named HPAACD1.3 and HPAA-CD6.1, respectively. The resulting polymers were characterized by FTIR measurements. Figure 1 gives the FTIR spectra of pure HPAA, HPAACD1.3, and HPAA-CD6.1. For pure HPAA, the broad N
H stretching vibration around 3283 cm
1 indicates the existence of many amino groups. The bands at 2946 and 2820 cm
1 correspond to the antisymmetric and symmetric
CH2
stretching vibration, respectively. The absorption peaks of amide groups are clearly observed at 1647 and 1538 cm
1, while the band at 1460 cm
1 results from the deformation vibration of the
CH2
groups. The FTIR spectral profile of HPAA-CD1.3 and HPAA-CD6.1 is quite similar to that of pure HPAA, suggesting that all polymers share the basic molecular structure. In the meantime, by comparison with pure HPAA, a new absorption at 1033 cm
1 appears in the curves of HPAA-CD1.3 and HPAA-CD6.1 and can be attributed to the C
O
C stretching vibrations of β-CDs. 1164

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Table 1. Reaction Conditions and Results of Michael Addition Copolymerization of MBA, AP, and EDA-β-CD monomer molar ratio entry

MBA (mmol)

AP (mmol)

EDA-β-CD (mmol)

actual CD content (%)a

Mnb (  104)

Mwb (  104)

Mw/Mnb

DBa

Tgc (°C)

HPAA

20

20

0

0

1.23

2.02

1.64

0.33

32.2

HPAA-CD1.3

20

19.5

0.5

1.3

0.95

1.72

1.81

0.34

38.1

HPAA-CD6.1

20

18

2

6.1

1.44

3.40

2.36

0.30

65.7

a

Actual CD content in the resultant polymers and the degree of branching (DB) were calculated from quantitative 13C NMR analysis. b Molecular weights and polydispersities were determined by GPC-MALLS. c Tg was measured by DSC.

Figure 1. FTIR spectra of pure HPAA and HPAA-CDs. Figure 3. Quantitative CDs.

13

C NMR spectra of pure HPAA and HPAA-

Scheme 2. Schematic Illustration of the Molecular Structure of HPAA-CDs

Figure 2. 1H NMR spectra of pure HPAA and HPAA-CDs.

With the increase of β-CD content, the peak intensity of 1033 cm
1 band increases. The NMR studies further confirmed the formation of HPAA-CDs. In the 1H NMR spectrum of pure HPAA (Figure 2), the signals at 2.1
2.7 ppm are attributed to the methylene groups in AP units, while the peak at 4.3 ppm comes from the protons of methylene groups between two amide groups in MBA units. After surface modification of HPAA by β-CDs, several new signals at 3.3
3.8, 4.8, and 5.7 ppm corresponding to the protons of β-CD appear. Figure 3 gives the quantitative 13C NMR spectra of HPAA, HPAA-CD1.3, and HPAA-CD6.1. For pure HPAA, the carbonyl peak is located at 173 ppm while all methylene signals are found in the high field region between 32 and 62 ppm. In the 13C NMR spectra of HPAA-CD1.3 and HPAA-CD6.1, the signals at 101, 81, 72, and 61 ppm are assigned to the methylene and methine groups of β-CD and become strong with the increase of β-CD content. Both FTIR

and NMR studies demonstrate that different amounts of β-CDs have been successfully incorporated onto the surface of HPAA. In order to calculate the degree of branching (DB) of pure HPAA and HPAA-CDs, all possible structure units including linear, branched, and terminal units are listed in Scheme 2. The two-dimensional NMR (2D-NMR) technique was used to assign the molecular structure.29,30 As one example, Figure 4 gives the 1 H
1H COSY and 13C
1H HSQC NMR spectra of HPAACD6.1. On the basis of the cross-peaks in the 2D-NMR spectra, the assignment of each structural unit for HPAA-CD6.1 is performed, and the corresponding details are given in Figure 4. 1165

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Figure 5. (a) UV
vis spectra and (b) fluorescence emission spectra of pure HPAA and HPAA-CDs in aqueous solutions (1 mg/mL) measured at room temperature, λex = 375 nm.

Figure 4. 2D NMR spectra of HPAA-CD6.1: (a) 1H
1H COSY spectrum, (b) 13C
1H HSQC spectrum.

Correspondingly, the DB values can be calculated according to the following equation:31 DB ¼ ðD þ TÞ=ðD þ T þ LÞ where D, T, and L represent the fractions of the branched, terminal, and linear units, respectively. As listed in Table 1, the DB values of polymerized samples are higher than 0.30, suggesting the formation of hyperbranched polymers. UV
vis and Fluorescent Properties of HPAA-CDs. Although without traditional fluorescent functional groups, both poly(amido amine) (PAMAM) dendrimer and hyperbranched poly(amido amine) (HPAA) are found to exhibit photoluminescence. It is believed that the key unit for the photoluminescence property of PAMAM dendrimer and HPAA is the tertiary amine with lone pair electrons inside the tight structure of highly branched polymers.17,20 However, the photoluminescence of these polymers is weak. In the present work, different amounts of β-CD were incorporated onto HPAA to form HPAA-CDs, which limited the molecular mobility and then improved the photoluminescence. The optical properties of HPAA-CDs were studied by UV
vis and fluorescence spectroscopy. Figure 5a gives the UV
vis spectra of different polymer aqueous solutions with

a concentration of 1 mg/mL. An obvious absorption band at 295 nm related to the tertiary nitrogen can be observed. This UV
vis absorption increases with the β-CD content, because of the improved molecular rigidity of the HPAA backbone. Figure 5b shows the corresponding fluorescence emission spectra. It can be found that the HPAA-CD displays a broad fluorescent band at ∼465 nm under excitation at 375 nm. With the increase of β-CD content from 0% to 6.1%, the fluorescence at 465 nm is enhanced significantly. The photoluminescence enhancement of HPAA-CDs may result from the restricted rotational motion of the terminal chains caused by the steric hindrance of big β-CD units, which decreases the collisional relaxation and fluorescence self-quenching.19 The quantum yields of HPAA, HPAA-CD1.3, HPAA-CD6.1, and NH2-terminated G4 PAMAM dendrimer were determined to be 0.59%, 0.68%, 0.73%, and 0.25%, respectively. Although the quantum yields of all the samples are not large, that of HPAA-CDs seem to be higher than that of G4 PAMAM dendrimer. The fluorescence emission spectra show that HPAA-CDs emit much stronger fluorescence than G4 PAMAM dendrimer with the same solution concentration. The experimental details are given in the Supporting Information. The strong fluorescence and cationic characteristics of HPAA-CD make it an excellent fluorescent nonviral gene delivery vector without fluorescent probes. Cytotoxicity of HPAA-CDs. It is very important to evaluate the potential toxicity of polymeric materials for biomedical applications. Here, the in vitro cytotoxicity of the pure HPAA and two HPAA-CD samples was evaluated using the MTT assay against COS-7 cells with PEI (Mw = 25 kDa) as a control. The MTT assay is based on the ability of a mitochondrial dehydrogenation enzyme in viable cells to cleave the tetrazolium rings of 1166

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Figure 6. Cell viability of COS-7 against PEI, pure HPAA, and HPAA-CDs after culturing for 24 h. Results are presented as the mean ( SD (n = 6).

Figure 7. Agarose gel electrophoretic images. For each polymer, gel electrophoresis assays were performed at the N/P ratios of 0, 5, 10, 20, 30, 40, 60, and 90 from left to right in each image. The left lane (N/P = 0) contains DNA alone (no polymer) and is used as a control in each image. (a
c): Agarose gel electrophoresis data of DNA released from the complex with pure HPAA, HPAA-CD1.,3 and HPAA-CD6.1, respectively.

the pale-yellow MTT and form formazan crystals with dark-blue color. Therefore, the number of surviving cells is directly proportional to the level of formed formazan.32,33 Figure 6 presents the cell viability after 24 h incubation with polymer samples at different concentrations. All the samples show cytotoxicity when the polymer concentration is above 50 μg/ mL. It has been well reported that the strong positive charge of primary amino groups increases the cytotoxicity significantly.34 Moreover, increasing molecular weight of polymers also induces enhanced cytotoxicity.35 Thus, the relatively high cytotoxicity of HPAA and its CD-based derivatives may be attributed to the strong positive charges of primary amines and the high molecular weight. In comparison to the PEI control, both pure HPAA and HPAA-CD samples exhibit less toxicity in COS-7 cells and the cytotoxicity decreases with the incorporation of β-CDs. β-CD has been demonstrated to be biodegradable, which is of great importance in reducing toxicity by integrating it into polycations.36 After the surface β-CD modification, a part of the primary amines of HPAA is replaced by the biodegradable and low cytotoxic β-CDs, resulting in the reduced cytotoxicity. Polymer/pDNA Complexes. As one of the promising biodegradable polymers, poly(amido amine) was applied as an efficient nonviral gene vector. Considering the similar structure, the possibility of photoluminescent HPAA-CDs to be used as nonviral gene vectors was also evaluated. First, the ability of HPAA-CDs to form complexes with pDNA was examined by agarose gel electrophoresis at various molar ratios of polymer nitrogen to pDNA phosphorus (N/P ratios). Figure 7 shows the gel retardation results of the HPAA-CD/pDNA complexes.

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Figure 8. Flow cytometry histogram profiles of COS-7 cells incubated with the HPAA-CD6.1/pDNA complexes.

It indicates that, when the N/P ratio is higher than 5, the migration of pDNA is retarded completely for pure HPAA, HPAACD1.3, and HPAA-CD6.1. The agarose gel electrophoresis results clearly demonstrate that HPAA-CDs can condense pDNA very well. Cell Internalization Studies. Benefiting from the strong photoluminescence of HPAA-CD, the cell internalization studies of HPAA-CD/pDNA complexes could be performed conveniently without fluorescent labeling. The cellular uptake of HPAA-CD6.1 which emitted the strongest fluorescence was determined by measuring the intracellular fluorescence intensity using flow cytometry. The HPAA-CD6.1/pDNA complexes (N/P = 30) were added to culture medium, and the COS-7 cells were incubated at 37 °C for the desired time. Here, COS-7 cells without any treatment were used as a blank control. Figure 8 gives the histograms of cell-associated HPAA-CD6.1 fluorescence. The relative geometrical mean fluorescence intensities of the HPAA-CD6.1/pDNA complex pretreated cells are much higher than that of non-pretreated cells. These prominent fluorescence signals are associated with the uptake of the HPAA-CD6.1/pDNA complexes into COS-7 cells. The mean fluorescence intensities almost do not change for incubation intervals longer than 30 min, indicating the rapid cellular internalization of the HPAA-CD6.1/pDNA complexes. To further confirm the cell permeability of the HPAA-CD6.1/ pDNA complexes, CLSM measurements were performed. In CLSM studies, the COS-7 cells were incubated with the HPAACD6.1/pDNA complexes for predesignated time (0.5 h, 2 h, 6 h, 10 h, and 24 h), and then the cell nucleus was stained with DAPI. The pretreated cells were observed under CLSM. Figure 9 shows that the HPAA-CD6.1 fluorescence appears in all cells but mainly in the cytoplasm. These results indicate that the HPAA-CD6.1/ pDNA complexes are successfully internalized by COS-7 cells and mainly resided in cytoplasm. Actually, this is very important for intracellular applications of the polymer complexes, such as intracellular gene delivery. In Vitro Transfection Assay. Considering the high DNA condensation ability and low cytotoxicity, HPAA-CDs can be used as an efficient nonviral vector for gene delivery. In vitro transfection activity of HPAA-CD/pDNA complexes was evaluated in COS-7 cells in DMEM supplemented with or without 10% FBS in comparison with PEI by the luciferase assay. All transfection efficiencies were obtained in the absence of chloroquine, a reagent known to disrupt the endosomal membrane 1167

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Figure 9. CLSM images of COS-7 cells incubated with HPAA-CD6.1/ pDNA complexes for predesignated time (from top to bottom: 0.5 h, 2 h, 6 h, 10 h, 24 h). The green fluorescence is from HPAA-CD6.1 (A, D, G, J, M), and the blue is from the nuclei (cell nuclei were stained with DAPI) (B, E, H, K, N). The third column illustrates overlaid images (C, F, I, L, O).

and to enhance transfection of DNA complexes by trafficking through the endolysosomal pathway. Figure 10a shows that HPAA-CD/pDNA complexes in the serum-free medium have similar transfection efficiencies at different N/P ratios, which are slightly smaller than that of the PEI control. The β-CDs seem to have no remarkable effect on the transfection efficiency in the system. For potential in vivo applications, the evaluation of transfection activity in the presence of serum is indispensible. Because the formation and stability of vector/pDNA complexes can be easily changed by the medium, many cationic vectors used for gene transfer experiments are no longer effective in the systems containing serum.37 In comparison to Figure 10a, Figure 10b shows that the transfection ability of HPAA-CD/ pDNA complexes decreases at low N/P ratios in the presence of 10% FBS. With the increase of N/P ratios from 10 to 60, the transfection efficiencies of HPAA-CD complexes improve and finally become quite close to that of the PEI control, indicating the high stability of HPAA-CD/pDNA complexes in the presence of serum. Complexation with Rhodamine B as Model Drug. It is wellknown that β-CD has important applications in pharmaceutical fields due to its strong ability to selectively form inclusion complexes with a wide range of guest molecules.38
44 For HPAA-CDs, the cavities of surface β-CDs can also be utilized to encapsulate drug molecules with proper size. Rhodamine B (RB) was chosen as a model drug. The obtained HPAA-CD/RB complexes were dissolved in water, and the fluorescent spectra

Figure 10. Transfection efficiency of pure HPAA and HPAA-CDs at various N/P ratios and branched PEI (25 kDa) at the optimal N/P ratio of 15 in COS-7 cells in different media: (a) serum free DMEM, (b) DMEM containing serum. Luciferase expression levels were measured 48 h later. Results are presented as the mean ( SD (n = 3).

Figure 11. Fluorescence emission spectra of free RB in aqueous solution (0.005 mg/mL) and RB-loaded HPAA-CDs in aqueous solutions(0.5 mg/mL), λex = 520 nm.

were acquired as shown in Figure 11. By comparison with the spectrum of free RB aqueous solution, the fluorescence of HPAA-CD/RB complexes shows an obvious blue shift from 1168

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Bioconjugate Chemistry 585 to 545 nm. With the increase of β-CD content, the fluorescence intensity increases rapidly. These observations demonstrate that RB has been successfully included into the cavity of β-CD. Therefore, the introduction of CD onto the surface of HPAA provides a novel photoluminescent delivery material in the combination of gene therapy and chemotherapy.

’ CONCLUSIONS Hyperbranched poly(amido amine)s (HPAAs) containing different amounts of β-CD (HPAA-CDs) have been synthesized by Michael addition copolymerization of MBA, AP, and EDA-βCD. In comparison with pure HPAA, HPAA-CDs show lower cytotoxicity and significantly enhanced photoluminescence. HPAA-CDs can condense pDNA very efficiently. The cell internalization of the HPAA-CD/pDNA complexes has been studied through flow cytometry and CLSM by detecting the fluorescence of HPAA-CD itself, which avoids the fluorescent labeling process. It demonstrates that the cellular uptake of HPAA-CD/pDNA complexes is very fast and HPAA-CDs mainly locate in the cytoplasm of the cells during the gene transportation process. At the same time, the inner cavities of β-CDs in HPAA-CDs can be used as drug containers. Thus, HPAA-CDs may have potential applications as delivery materials in the combination of gene therapy and chemotherapy. ’ ASSOCIATED CONTENT

bS

Supporting Information. Quantum yields and fluorescence emission spectra of pure HPAA, HPAA-CDs, and NH2terminated G4 PAMAM dendrimer in aqueous solutions. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel.: þ86-21-34205699; Fax: þ86-21-34205722; E-mail: xyzhu@ sjtu.edu.cn (X. Z.). Tel.: þ86-21-62232274; Fax: þ86-2154344800; E-mail: [email protected] (Q.C.).

’ ACKNOWLEDGMENT This work is sponsored by the National Natural Science Foundation of China (20974062) and National Basic Research Program 2009CB930400, the Fok Ying Tung Education Foundation (111048), Shuguang Program (08SG14), Shanghai Leading Academic Discipline Project (Project Number: B202), and China National Funds for Distinguished Young Scientists (21025417). ’ REFERENCES (1) Mintzer, M. A., and Simanek, E. E. (2009) Nonviral vectors for gene delivery. Chem. Rev. 109, 259–302. (2) Park, T. G., Jeong, J. H., and Kim, S. W. (2006) Current status of polymeric gene delivery systems. Adv. Drug Delivery Rev. 58, 467–486. (3) Li, S. D., and Huang, L. (2007) Non-viral is superior to viral gene delivery. J. Controlled Release 23, 181–183. (4) De Smedt, S. C., Demeester, J., and Hennink, W. E. (2000) Cation polymer based gene delivery systems. Pharm. Res. 17, 113–126. (5) Thomas, M., and Klibanov, A. M. (2003) Non-viral gene therapy: polycation-mediated DNA delivery. Appl. Microbiol. Biotechnol. 62, 27–34.

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