Design and Synthesis of Cationic Drug Carriers Based on

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Design and Synthesis of Cationic Drug Carriers Based on Hyperbranched Poly(amine-ester)s Yan Pang,† Qi Zhu,† Jinyao Liu,† Jieli Wu,‡ Ruibin Wang,‡ Suyun Chen,§ Xinyuan Zhu,*,†,‡ Deyue Yan,† Wei Huang,† and Bangshang Zhu*,‡ School of Chemistry and Chemical Engineering and Instrumental Analysis Center, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China, and Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, 197 Ruijin second Road, Shanghai 200025, P. R. China Received October 3, 2009; Revised Manuscript Received February 5, 2010

Novel cationic drug carriers based on hyperbranched poly(amine-ester)s were successfully prepared through protontransfer polymerization. Both vinyl and epoxy groups of commercially available glycidyl methacrylate monomer could be polymerized through oxyanionic initiation of triethanolamine in the presence of potassium hydride catalysis. By changing the molar ratios of triethanolamine/glycidyl methacrylate or potassium hydride/triethanolamine, we obtained a series of hyperbranched poly(amine-ester)s. The generation of highly branched poly(amine-ester)s was confirmed by 13C DEPT-135 NMR and 2D NMR techniques, and their degrees of branching were found to be 0.47 to 0.68. The structure and properties of hyperbranched poly(amine-ester)s were analyzed by dynamic light scattering, gel permeation chromatography, Fourier transformed infrared, differential scanning calorimeter, and ζ-potential measurements. Methyl tetrazolium (MTT) assay suggested that the cell viability after 48 h incubation with hyperbranched poly(amine-ester) concentrations up to 1 mg/mL remained nearly 100% compared with the untreated cells. The high cellular uptake of these cationic polymers was confirmed by flow cytometry and confocal laser scanning microscopy. Furthermore, conjugation of a model hydrophobic anticancer drug chlorambucil to hyperbranched poly(amine-ester)s inhibited the proliferation of MCF-7 breast cancer cells. MTT assay indicated that the chlorambucil dose required for 50% cellular growth inhibition against MCF-7 cells was 120 µg/mL. All of these results show that hyperbranched poly(amine-ester)s are promising materials for drug delivery.

Introduction Polycationic drug delivery systems have attracted much attention because of their unique characteristics, such as good water solubility and high cellular uptake efficiency.1,2 Many linear polycations, including polyamines, amino-functionalized polymers, and peptides with basic amino acid residues have been widely used as drug carriers.3-6 Different from the conventional linear polymer, the dendritic polymer has a globular molecular structure and plenty of functional end groups as well as good solubility, which makes it a promising carrier for drug delivery.7-9 In recent years, various dendritic polycations including dendrimers and hyperbranched polymers have been used to construct different drug delivery systems.10-18 Moreover, thanks to the existence of many functional terminals, the drug molecules, target groups, and fluorescent agents can be readily conjugated onto the surface of a same dendritic polymer so that multiobjectives such as treating, targeting, and diagnosis can be achieved simultaneously.19 Unfortunately, most dendritic polycations show significant cytotoxicity, mainly associated with the strong positive charge and nondegradability.20-22 Therefore, recent development of the polycationic carrier systems has been focused on the enhancement of their biocompatibility. It has been suggested that the biocompatibility could be improved through the introduction of water-soluble and biocompatible * To whom correspondence should be addressed. Tel: +86-21-34205699. Fax:+86-21-34205722.E-mail:[email protected](X.Z.);[email protected] (B.Z.). † School of Chemistry and Chemical Engineering. ‡ Instrumental Analysis Center. § Department of Nuclear Medicine, Ruijin Hospital, School of Medicine.

surface groups or anionic charges. For example, poly(ethylene glycol) chains or oligosaccharide units have been frequently used for modification.23-30 However, these surface modifications usually lead to the change of inherent properties because the primary parameters of dendritic polymers such as solubility, charge distribution, or receptor-mediated interaction strongly depend on the nature of the surface groups.24 Therefore, the design and synthesis of dendritic polycations with low toxicity are still required. It has been well reported that biodegradable cationic polymers are potential drug carriers with low toxicity.31-35 In the present work, we designed and synthesized a novel kind of hyperbranched poly(amine-ester)s by proton-transfer polymerization of commercial glycidyl methacrylate (GMA) and triethanolamine (TEOA). The ester bonds in the backbone made the polymer biodegradable, and the amine groups in the polymer made it highly cellular permeable. Different molar ratios of TEOA/GMA or KH/TEOA were used to prepare different hyperbranched polymers. In vitro evaluation of hyperbranched poly(amine-ester)s through methyl tetrazolium (MTT) assay, flow cytometry, and confocal laser scanning microscopy (CLSM) confirmed the low toxicity and high cellular uptake of these cationic drug carriers. Additionally, a model anticancer drug chlorambucil was conjugated to hyperbranched poly(amineester)s, which exhibited the growth inhibition against MCF-7 cells.

Experimental Section Materials. Potassium hydride (KH, 30 wt % dispersion in mineral oil, Acros), N-hydroxysuccinamide (NHS, Acros), N,N-dicyclohexyl-

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Table 1. Reaction Conditions and Results of Proton-Transfer Polymerization of TEOA and GMA entrya

T/Gb

KH/Tc

diameter (nm)d

Mw (×103)e

Mn (×103)e

Mw/Mne

DBf

Tg (°C)g

ζ-potential (mV)

P1 P2 P3 P4 P5 P6

1:1 1.1:1 1.3:1 1.5:1 1:1 1:1

1:5 1:5 1:5 1:5 1:2 1:3

3.1 2.3 2.4 2.1 3.1 3.4

8.2 9.4 8.5 8.1 9.1 9.5

5.2 6.5 5.6 5.1 6.3 6.8

1.58 1.44 1.51 1.59 1.45 1.40

0.48 0.52 0.68 0.62 0.47 0.50

-47 -40 -32 -41 -32 -29

2.6 ( 0.4 3.5 ( 0.2 5.5 ( 0.7 7.1 ( 0.3 2.2 ( 0.6 2.1 ( 0.4

a Polymer obtained from the corresponding condition listed. b Molar ratio of monomer TEOA to GMA. c Molar ratio of KH to TEOA. d Determined by DLS. e Molecular weights and polydispersity were determined by GPC. f Degree of branching (DB) was calculated from quantitative 13C NMR analysis. g Determined by DSC.

carbodiimide (DCC, Aldrich), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Aldrich), chlorambucil (Aldrich), 4-dimethylamino-pyridine (DMAP, Aldrich), and polyethyleneimine (PEI, water free, Mw ) 25 kDa, Mn ) 10 kDa, Aldrich) were used as received. TEOA was distilled under reduced pressure. GMA (Acros), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF) were dried over calcium hydride (CaH2) and distilled under reduced pressure. Tetrahydrofuran (THF) was dried by refluxing over fresh sodiumbenzophenone complex (a deep purple color indicating an oxygen- and moisture-free solvent) and distilled just before polymerization. Clear polystyrene tissue-culture-treated 6-well and 96-well plates were obtained from Corning Costar. Other reagents were purchased from Shanghai Chemical Reagent Co. and used as received. Synthesis of Hyperbranched Poly(amine-ester)s. The cationic hyperbranched poly(amine-ester)s were prepared by proton-transfer polymerization. A typical polymerization procedure is as follows: A suspension of KH in mineral oil (30% in weight) was introduced in a dry preweighted 100 mL Schlenk flask under argon (Ar). The mineral oil was removed by three extractions with THF, and the remaining THF was removed by vacuum. When KH was completely dried, the flask was weighted again to determine the amount of KH (0.698 g, 17.4 mmol). Then, 40 mL of DMSO and TEOA (12.9 g, 86.5 mmol) was introduced to the flask. The solution was stirred for 30 min to form the potassium alcoholate. Subsequently, GMA (11.8 g, 83.0 mmol) was added by syringe, and the polymerization was conducted at 80 °C for 48 h. Upon completion of the polymerization, the mixture was precipitated in 1000 mL of acetone/diethyl ether (v/v 1/4). The product was redissolved in methanol and neutralized by filtration over cationexchange resin. The obtained polymer was precipitated twice from methanol solution in cold diethyl ether and then dried in vacuo at 25 °C for 24 h. Similarly, polymers with different dendritic structures were synthesized by changed TEOA/GMA or KH/TEOA molar ratios. (See Table 1.) The resultant purified product was highly viscous. Rhodamine-B-Conjugated Polymers. A typical rhodamine B (RB) conjugation reaction is described as follows: NHS (30.0 mg, 0.260 mmol) and RB (120 mg, 0.250 mmol) were dissolved in 15 mL of DMF, and then DCC (62.0 mg, 0.300 mmol) and DMAP (31.0 mg, 0.250 mmol) were added to the solution. The mixture was stirred at room temperature for 30 min. Hyperbranched poly(amine-ester) (P1, 360 mg, 0.0530 mmol) in 15 mL of DMF was added to the reaction solution. The reaction was conducted at room temperature for 48 h. Then, the insoluble substance was filtered, and DMF was removed by rotary evaporation. The residues were dissolved in distilled water. Exhaustive dialysis against exchanged distilled water was carried out for 4 days, followed by lyophilization to give the RB-conjugated polymer. Chlorambucil-Conjugated Polymers. In a typical experiment, chlorambucil (85.8 mg, 0.282 mmol), hyperbranched poly(amine-ester) (P1, 415 mg, 0.0732 mmol), DCC (64.0 mg, 0.310 mmol) and DMAP (3.44 mg, 0.0282 mmol) were mixed in a 100 mL flask with 30 mL DMF. The solution was stirred for 48 h at room temperature under a nitrogen atmosphere. Then, the insoluble dicyclohexylurea was filtered and DMF was removed by rotary evaporation. The crude product was purified by being repeatedly dissolved in methanol and then precipitated in diethyl ether. After being dried in vacuo at room temperature for 24 h, the chlorambucil-conjugated polymer was obtained.

Characterization of Polymers. 1H and 13C NMR spectra of the polymers were recorded using Varian Mercury Plus 400 MHz spectrometer with dimethyl sulfoxide-d6 (DMSO-d6) as a solvent. Quantitative 13C NMR spectra were measured by the method of inverse gated 1H decoupling. In DEPT experiments, the 1H tip angle θ was set to 135° to determine carbon multiplicities with CH, CH3 up, and CH2 down. 1H,1H-COSY, and 13C,1H-HSQC spectra were recorded using the standard pulse sequence provided by Varian. Fourier transformed infrared (FTIR) measurements were performed on a Bruker Equinox55 FTIR spectrometer with a disk of KBr. Dynamic light scattering (DLS) was performed on a ZETASIZER Nano-ZS90 apparatus. The molecular weights and molecular weight distributions were determined by gel permeation chromatography (GPC). GPC was performed on a Perkin-Elmer series 200 system (10 µm PL gel 300 × 7.5 mm mixed-B and mixed-C column, polystyrene calibration) equipped with a refractive index (RI) detector. DMF containing 0.01 mol/L lithium bromide was used as the mobile phase at a flow rate of 1 mL/min at 70 °C. The glass-transition temperature (Tg) of hyperbranched polymers was measured with a Perkin-Elmer Pyris 1 differential scanning calorimeter (DSC) under a nitrogen (N2) atmosphere. All samples were heated from -80 to 100 °C at a heating rate of 10 °C/min. The midpoint of the slope change of the heat capacity plot of the second heating scan was taken as Tg. For ζ-potential, the hyperbranched poly(amine-ester)s were dissolved in phosphate-buffered saline (PBS) (pH 7.2) at a concentration of 1 mg/mL. The ζ-potential was measured with Zetasizer 2000 (Malvern, U.K.). Values of ζ-potential were averaged over three repeated measurements. Cell Cultures. NIH 3T3 cells (a mouse embryonic fibroblast cell line) and MCF-7 cells (a human breast adenocarcinoma cell line) were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplied with 10% FBS (fetal bovine serum), and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C under a humidified atmosphere containing 5% CO2. Cytotoxicity. For cytotoxicity, the P1-P6 samples were dissolved in DMEM at diluted polymer concentrations from 0.001 to 10 mg/mL, respectively. NIH 3T3 cells were seeded in 96-well plates at an initial seeding density of 8.0 × 103 cells/well in 200 µL medium. After 24 h of incubation, the culture medium was removed and replaced with 200 µL of medium containing serial dilutions of poly(amine-ester)s. The cells were grown for another 48 h. Then, 20 µL of 5 mg/mL MTT assays stock solution in PBS was added to each well. After the cells were incubated for 4 h, the medium containing unreacted dye was removed carefully. The obtained blue formazan crystals were dissolved in 200 µL per well DMSO, and the absorbance was measured in a BioTek Elx800 at a wavelength of 490 nm. Cell Internalization. Cell internalization was characterized by flow cytometry and CLSM. Flow cytometry was used to provide statistics on the uptake of poly(amine-ester) into MCF-7 cells. MCF-7 cells (5.0 × 105 cells per well) were seeded in a 6-well tissue culture plate. After 24 h of culture, the RB-conjugated P1 dissolved in DMEM culture medium with a polymer concentration of 1 mg/mL was 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 media, rinsing with PBS buffer, and treating with trypsin. Data for 1.0 × 104 gated events

Design and Synthesis of Cationic Drug Carriers were collected, and analysis was performed by means of a BD FACSCalibur flow cytometer and CELLQuest software. For CLSM, MCF-7 cells (2.0 × 105 cells per well) were seeded on coverslips in a 6-well tissue culture plate. After 24 h of culture, the RB-conjugated P1 dissolved in DMEM culture medium with a polymer concentration of 1 mg/mL was added to different wells, and the cells were incubated at 37 °C for predetermined time intervals. After being washed with PBS, the cells were fixed with 4% formaldehyde for 30 min at room temperature, and the slides were rinsed with PBS three times. Finally, the slides were mounted and observed with a LSM510 META. Drug Delivery Property. The inhibition of chlorambucil-conjugated polymers against MCF-7 cells was evaluated in vitro by MTT assay using chlorambucil dissolved in DMSO as the control. (The final concentration of DMSO in medium was 0.5% v/v.) MCF-7 cells were seeded in 96-well plates at an initial seeding density of 8.0 × 103 cells/ well in 200 µL medium. After 24 h of incubation, the culture medium was removed and replaced with 200 µL of medium containing serial concentrations of P1-conjugated chlorambucil from 1 to 400 µg/mL. The cells were grown for another 96 h. Then, 20 µL of 5 mg/mL MTT assays stock solution in PBS was added to each well. After the cells Scheme 1. Synthesis of Hyperbranched Poly(amine-ester)s


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were incubated for 4 h, the medium containing unreacted MTT was carefully removed. The obtained blue formazan crystals were dissolved in 200 µL per well DMSO, and the absorbance was measured in a BioTek Elx800 at a wavelength of 490 nm. The cytotoxicity of hyperbranched poly(amine-ester)s against MCF-7 cells was measured by the same method.

Results and Discussion Synthesis and Characterization of Hyperbranched Poly(amine-ester)s. The dendritic polycationic drug carriers have been prepared by different polymerization methods, and here the proton-transfer polymerization was used for the first time to synthesize the cationic dendritic polymers. GMA is a typical monomer for proton-transfer polymerization because of the existence of two different reactive groups, namely, a vinyl group and an epoxy group.36-40 Both the epoxy group and the double bond of GMA could be polymerized through oxyanionic initiation.41 To obtain the polycationic carriers, TEOA, a triol

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Figure 1. Various structural units of hyperbranched poly(amineester)s.

monomer with a nitrogen atom, was chosen to initiate the proton-transfer polymerization of GMA with the help of KH catalysis. The synthesis route of cationic hyperbranched poly(amineester)s from TEOA and GMA through proton-transfer polymerization is shown in Scheme 1. During the polymerization, the TEOA 1 first reacts with potassium hydride to originate initiator 2. Because of the bifunctionality of GMA, there are two possibilities for the reaction of 2 with GMA. 2 initiates the vinyl group of GMA leading to 3, whereas 2 reacts with the epoxy group of GMA producing 4. Proton-transfer from the secondary alkoxide or carbonanion to the more stable primary alkoxide is expected, thus producing 5, 6, and 7.42-45 3, 4, 5, 6, and 7 each contain a polymerizable group (vinyl or epoxy group) and an anionic center and can be considered to be inimers (initiatormonomer).46 Further polymerization of these inimers gives rise to the hyperbranched species with both oxyanions and carbanions. Finally, the hyperbranched polymer 8 forms. When the polymerization was carried out in DMSO at 80 °C for 48 h, a highly viscous product was obtained. The reaction proceeded homogeneously, and no gelation took place throughout the polymerization. Different molar ratios of TEOA/GMA or KH/ TEOA were designed to prepare different hyperbranched poly(amine-ester)s. The experimental conditions and corresponding characterization data are summarized in Table 1. Careful examination of the structure of polymer 8 reveals that three terminal subunits, five linear subunits, and five dendritic subunits may be present. Figure 1 displays the structural units of hyperbranched poly(amine-ester)s synthesized from TEOA and GMA. A DEPT-135 spectrum was recorded to distinguish methylene and methane or methine carbons of poly(amine-ester) (Figure 2). According to the DEPT spectrum, the peaks 5, 12, 15, 24, and 27 are assigned to the methyl groups from GMA units. The double-bond signals and epoxide resonances are observed at peaks 6, 7, and 9, respectively. The peaks 25 and 28 are attributed to quaternary carbon atoms, which disappear in the corresponding DEPT spectra. The branched topology of polymerized product could be verified by 2D-NMR analysis.41,47-49 The 1H,1H-COSY (Figure 3A), and 13C,1H-

Figure 2. 13C DEPT-135 NMR spectrum of hyperbranched poly(amineester) synthesized from TEOA and GMA in d6-DMSO. (A and B are the same 13C DEPT-135 NMR spectrum with different regions from 190 to 0 and 85 to 5 ppm, respectively).

HSQC spectra (Figure 3B) were performed for further assignment of the peaks. The signals in Figure 3A reveal that 1-H/ 2-H, 3-H/4-H, 5-H/7-H, 7-H/7-H, 11-H/13-H, 12-H/13-H, 15H/16-H, 21-H/22-H, and 33-H/34-H were in different spin systems. On the basis of the information of the 1H,1H-COSY and 13C,1H-HSQC spectra, the detailed assignment is shown in Figure 3. Correspondingly, the degree of branching (DB) for the hyperbranched poly(amine-ester) could be calculated according to the following equation50

DB ) (D + T)/(D + T + L) where D, T, and L represent the fractions of the dendritic, terminal, and linear units, respectively. The DBs of the samples synthesized from TEOA and GMA are listed in Table 1, which confirms the formation of highly branched products. FTIR results provide the chemical structure information of hyperbranched poly(amine-ester)s. Figure 4 gives a representative FTIR spectrum of sample P1 in Table 1. The absorption peaks of C-N-C and C-O-C stretching vibrations are clearly observed at 1118 and 1050 cm-1, respectively. The band at 1458 cm-1 results from the asymmetric deformation vibration of the -CH2- groups. A strong ester carbonyl band at 1730 cm-1 confirms the presence of ester bonds. The bands at 2872 and 2944 cm-1 correspond to the symmetric and asymmetric -CH3 stretching vibration, whereas the broad O-H stretching vibration around 3374 cm-1 indicates the appearance of many hydroxyl

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Figure 5. Cytotoxicity of polymers against NIH 3T3 cells of different polymers compared with control (PEI).

Figure 3. 2D NMR spectra of poly(amine-ester): (A) 1H,1H-COSY spectrum and (B) 13C,1H-HSQC spectrum.

Figure 4. Representative FTIR spectrum of hyperbranched poly(amineester) (P1).

groups. Additionally, GPC, DSC, and ζ-potential analyses were further used to measure the structure and properties of hyperbranched poly(amine-ester)s in details (Table 1). GPC measurements suggest that the weight-average molecular weights of the polymers are around 9.0 × 103, with PDI about 1.5. DSC measurements show that the glass-transition temperature (Tg) of the obtained polymers ranges from -47 to -29 °C, further confirming the highly branched structures of poly(amine-ester)s.

The introduction of flexible units such as ether bond into the macromolecular structure may plasticize the polymers, which reduces Tg of hyperbranched polymers in comparison with that (78 °C) of linear poly(glycidyl methacrylate).41 ζ-Potential analysis demonstrates that the hyperbranched poly(amine-ester)s are positively charged, attributing to the presence of numerous tertiary amines. Cell Cytotoxicity. The cytotoxicity of dendritic polycations is usually high, and thus it is necessary to evaluate the potential toxicity of the hyperbranched poly(amine-ester)s for drug delivery application. Here, in vitro cytotoxicity of the obtained hyperbranched poly(amine-ester)s with different TEOA/GMA molar ratio against NIH 3T3 cells was studied through MTT assay with the PEI 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 the pale yellow MTT and form formazan crystals with a dark blue color. Therefore, the number of surviving cells is directly proportional to the level of formed formazan.51,52 As shown in Figure 5, cells cannot tolerate the treatment with PEI at only 0.01 mg/mL. In contrast, the cell viability after 48 h of incubation with hyperbranched poly(amine-ester)s up to 1 mg/mL remains nearly 100% compared with the untreated cells. A slight decrease in NIH 3T3 cell viability is observed when the polymer concentration is >1 mg/mL. Even when the polymer concentration reaches 10 mg/mL, the cell viability is still ∼80%, suggesting the low cytotoxicity of hyperbranched poly(amine-ester)s. It has been well reported that the strong positive charge of primary amino groups increases the cytotoxicity significantly,53 whereas the introduction of biodegradable ester groups into polymer backbone decreases the cytotoxicity dramatically.35 Therefore, the low cytotoxicity of hyperbranched poly(amine-ester)s might be attributed to the absence of primary amino groups and the presence of biodegradable ester bonds. Cell Internalization Studies. Hyperbranched poly(amineester)s have a ζ-potential value ranging from 2.1 to 7.1 mV (Table 1), so the positive charge of hyperbranched poly(amineester)s enhances the electrostatic interaction between the polymers and negatively charged cell membranes. Flow cytometry analysis was performed to test cellular uptake of hyperbranched poly(amine-ester)s by MCF-7 cells. MCF-7 cells were cultured for predetermined time intervals with polymer concentration of 1 mg/mL before analysis (polymer was labeled with RB, see Scheme 2). Cellular uptake was determined as the number of fluorescent cells increased. Histograms of cellassociated RB fluorescence for MCF-7 cells are shown in Figure 6. The relative geometrical mean fluorescence intensities of polymer-pretreated cells are about 15-30 times those of non-

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Scheme 2. Synthesis of Hyperbranched Poly(amine-ester)s Conjugates

pretreated cells. The fast enhancement of fluorescence signals indicates high cellular uptake of hyperbranched poly(amineester) by MCF-7 cells. It is considered that the cellular uptake efficiency of polycations is presumably related to a cationic surface property. Consequently, the increased binding is followed by internalization within the cells through adsorptive endocytosis.1,20 The cellular uptake of hyperbranched poly(amine-ester)s by MCF-7 cells was further evaluated by CLSM. MCF-7 cells were incubated with hyperbranched poly(amine-ester) (1 mg/mL) at 37 °C for 1 h. Subsequently, the cell nucleus was stained by 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and then fixed with formaldehyde. RB fluorescence of the cells was observed directly under CLSM. As shown in Figure 7, for the polymer-pretreated cells with 1 h of incubation, detectable RB fluorescence is observed mainly in the perinuclear region instead of the nucleus. This result indicates that the cellular uptake of the hyperbranched poly(amine-ester)s is based on a endocytosis mechanism.7 Unlike the simple passive diffusion

Figure 6. Flow cytometry histogram profiles of MCF-7 cells incubated with RB-labeled hyperbranched poly(amine-ester) P1 at different intervals.

Figure 7. CLSM images of MCF-7 cells incubated with RB-labeled hyperbranched poly(amine-ester) P1 for 1 h (Cell nucleuses were stained with DAPI).

of small molecules between the extracellular and intracellular milieu, the cellular uptake of hyperbranched poly(amine-ester)s by endocytosis processes is unidirectional.54 Therefore, hyperbranched poly(amine-ester)s are efficiently transported. Drug Conjugate and Cell Viability Analysis. The good biocompatibility and high cellular uptake efficiency of these cationic hyperbranched poly(amine-ester)s encouraged us to evaluate further their potential as a polymeric drug carrier. Chlorambucil, an aromatic nitrogen mustard, was used as a model hydrophobic anticancer drug. The chlorambucil-conjugated polymers were prepared by coupling the end hydroxyl groups of hyperbranched poly(amine-ester)s with chlorambucil in the presence of DCC and DMAP in dry DMF (Scheme 2). The resulting conjugate was then purified by being repeatedly dissolved in methanol and precipitated in diethyl ether. NMR analysis was performed to evaluate the drug content of the final obtained conjugate. Calculating from the 1H NMR spectrum of conjugate, the chlorambucil content was determined to be 7.06 wt %. To demonstrate the therapeutic potential of the obtained conjugate, in vitro evaluation was performed by MTT assay against an MCF-7 breast cancer cell line. First, the toxicity of pure hyperbranched poly(amine-ether) against MCF-7 cells was investigated, and the MTT results confirmed the low cytotoxicity of polymer carriers again. (See Figure S3 in the Supporting Information.) To compare the cytotoxic activity of the chlorambucil-conjugated polymer with that of the free drug, chlorambucil in free form was used as a control. The MCF-7 cell proliferation results are shown in Figure 8. It is found that the conjugated chlorambucil dose required for 50% cellular growth inhibition (IC50) is 120 µg/mL. This result indicates that the conjugate is able to enter the cell and produce the desired pharmacological action. The IC50 of free chlorambucil is 50 µg/ mL, which exhibits higher inhibition compared with conjugated chlorambucil. This is a significant result because the conjugated chlorambucil is less toxic than the free drug. It has been well documented that the free drug is more potent than the conjugated drug.7 The antiproliferation effect of the chlorambucil-conju-

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50633010) and National Basic Research Program 2009CB930400, the Fok Ying Tung Education Foundation (111048), Shuguang Program (08SG14), and Shanghai Leading Academic Discipline Project (project number: B202). Supporting Information Available. Experimental details, representative 1H NMR spectra of P1 degradation in D2O, CLSM images of MCF-7 cells incubated with RB-labeled hyperbranched poly(amine-ester) P4, and cytotoxicity of P1 against MCF-7 cells at different concentrations. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes

Figure 8. Activity of chlorambucil-conjugated hyperbranched poly(amine-ester) against MCF-7 cells.

gated hyperbranched poly(amine-ester) is most likely related to the drug release from the conjugate in the course of incubation. Once the conjugate enters the cell, it is conceivable that the acidic pH and the enzymes in the endosomes would eventually hydrolyze the ester bond and slowly release the drug, which provides the therapeutic effect.8

Conclusions The proton-transfer polymerization has been successfully extended to prepare novel cationic hyperbranched poly(amineester)s. The commercially available GMA monomer has two different reactive groups, namely, a vinyl group and an epoxy group. Both the epoxy group and the double bond of GMA can be polymerized through oxyanionic initiation of cationic triol TEOA in the presence of KH catalysis. Different molar ratios of TEOA/GMA or potassium hydride/TEOA were investigated. The highly branched architecture of polymerized products has been confirmed by a 2D NMR technique, and the structure and properties of the resulting hyperbranched poly(amine-ester)s have been systematically analyzed by FTIR, DLS, GPC, DSC, and ζ-potential measurements. The cytotoxicity of hyperbranched poly(amine-ester)s has been evaluated by MTT assay against NIH 3T3 cells. Compared with the untreated cells, the cell viability after 48 h of incubation with polymers up to 1 mg/mL remains nearly 100%, indicating the low toxicity of this novel kind of cationic hyperbranched polymers. Flow cytometry and CLSM analysis suggests that the polycationic carriers can be easily internalized by MCF-7 cells and preferentially accumulated in the cytoplasm instead of the nucleus. Finally, chlorambucil as a model hydrophobic anticancer drug has been conjugated onto the surface of hyperbranched poly(amineester)s. IC50 of the chlorambucil-conjugated polymer is found to be 120 µg/mL using MTT assay against MCF-7 cells in vitro. Benefiting from the plenty of hydroxyl terminals of hyperbranched poly(amine-ester)s, these drug conjugates could be further functionalized with a targeting moiety to deliver the drugs to specific cells in vivo. All of these results indicate that hyperbranched poly(amine-ester) could provide opportunities for the design of drug delivery systems and therapeutic applications. Acknowledgment. This work is sponsored by the National Natural Science Foundation of China (20974062, 50773037,

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BM100007S

Design and Synthesis of Cationic Drug Carriers Based on

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