Reducing the Cytotoxity of Poly(amidoamine ... - ACS Publications

Jun 18, 2013 - Yan Wang , Lina Li , Junjie Li , Boguang Yang , Changyong Wang , Wancai ... Qinghua Yang , Wenchen Li , Longgang Wang , Guangzhi Wang ...
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Reducing the Cytotoxity of Poly(amidoamine) Dendrimers by Modification of a Single Layer of Carboxybetaine Longgang Wang, Zhen Wang, Guanglong Ma, Weifeng Lin, and Shengfu Chen* State Key Laboratory of Chemical Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, People’s Republic of China ABSTRACT: The surface primary amines of generation five poly(amido amine) (G5 PAMAM) dendrimer were modified by different amounts of carboxybetaine acrylamide (CBAA). As a result, the fully modified molecules (CBAA-PAMAM-20, obtained from the 20:1 molar ratio of CBAA molecules to amino groups in modification solution) show excellent compatibility with protein and cells. CBAA-PAMAM-20 and fibrinogen (Fg) could coexist in solution without forming aggregation, indicating very weak interaction force between CBAA-PAMAM-20 and fibrinogen. CBAA-PAMAM-20 exhibits almost undetectable hemolytic activity, while other partially modified ones cause severe hemolysis and fibrinogen aggregation. Furthermore, the membrane of human umbilical vascular endothelial cell (HUVEC) remains intact after 24 h incubation with CBAA-PAMAM-20. The cytotoxicity assay of HUVEC cells and KB cells also showed that the CBAAPAMAM-20 was not cytotoxic up to a 2 mg/mL concentration (>90% cell viability). In short, a thin compact layer of zwitterionic carboxybetaine could reduce the cytotoxicity of PAMAM through minimizing the interaction with protein and cell membranes, which suggest that the carboxybetaine-coated PAMAM could be a useful platform for biocompatible carriers to load contrast agents and drugs.

1. INTRODUCTION The high level of control over the architecture of dendrimers, their size, shape, branching length, density and their surface functionality makes these compounds ideal carriers in biomedical applications such as drug delivery, gene transfection, and imaging.1 There are a large number of dendrimeric structures that have been reported in the literature, among them, the commercially available polyamidoamine is one of the most widely explored dendrimers for biomaterial applications.2 However, as has been widely documented, dendrimers bearing NH2 termini display concentration- and generation-dependent cytotoxicity.3 The low biocompatibility of PAMAM in blood is one key obstacle in various applications of dendrimer. Recent results showed that cationic generation 7 PAMAM dendrimers could induce fibrinogen (Fg) aggregation in a thrombinindependent manner to generate dense, high-molecular-weight Fg aggregates with minimal fibrin fibril formation.4 All of these restrict the PAMAM in biomedical applications. It is believed that the cytotoxicity might be caused by the interactions between positively charged dendrimers and negatively charged molecules located on the cell membrane.5 It was also reported that the dendrimer cytotoxicity is dependent on the chemistry of the core but is most strongly influenced by the nature of the dendrimer surface.3 Thus, various molecules, such as poly(ethylene glycol)(PEG),6,7 acetyl groups,8,9 lauroyl groups,10 dimethyl itaconate,11 or phosphorylcholine (PC),5 have been used to modify the cationic PAMAM dendrimers to reduce cytotoxcity since these © 2013 American Chemical Society

modification could likely neutralize and shield the positive charge on the dendrimer surfaces through changing primary amine groups to an amide bond. PEG is currently believed to be the most prevalent material to increase the biocompatibility of PAMAM dendrimers due to the excellent biocompatibility, which is mainly related with its high resistance to nonspecific protein adsorption. In fact, polyzwitterionic materials, such as poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC),12,13 poly(sulfobetaine methacrylate) (pSBMA), 14,15 poly(carboxybetaine methacrylate) (pCBMA), 1 6, 17 poly(carboxybetaine acrylamide) (polyCBAA),18,19 or simply mixed charge materials,20 have been recognized as more stable and effective ones than PEG to resist nonspecific protein adsorption. Only a single layer of zwitterionic groups could effectively resist nonspecific protein adsorption.21 Thus, it is possible to enhance biocompatibility of the PAMAM dendrimers through surface modification by zwitterionic materials. Although PC partially modified PAMAM was investigated,5 it is important to know whether the fully modified PAMAM could form a rigid zwitterionic shell and further reduce the interaction with protein molecules and cells, which might meet the challenge of biocompatibility in blood, a highly complicated biological solution, when the modified PAMAM is used as a carrier of contrast agents and drugs. Received: February 17, 2013 Revised: May 29, 2013 Published: June 18, 2013 8914

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control and PBS as the negative control. The percent of hemolysis was calculated as follows

Therefore, zwitterionic CBAA is used to modify the PAMAM dendrimers with different degrees to investigate their biocompatibility in this work.

hemolysis% = [(sample absorbance − negative control) /(positive control − negative control)] × 100%

2. MATERIALS AND METHODS 2.1. Materials. RPMI 1640 medium, Trypsin 0.25%, and fetal bovine serum (FBS) were purchased from Sijiqing Biological Engineering Materials Co., Ltd. The dialysis bag (MWCO = 14 000) was purchased from Spectrum Laboratories Inc. Vivaspin 500 ultrafiltration tube poly(ether sulfones) membrane (MWCO = 10 000) was acquired from Sartorius. Methanol, dimethyl sulfoxide, sodium chloride, acetone, and ether were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. Amine-terminated PAMAM dendrimers of generation 5 (G5 PAMAM) with ethylenediamine core in 5% methanol solution and Fg from bovine were purchased from Sigma-Aldrich. N-[3-(Dimethylamino)propyl] acrylamide (DMAPA, 98%) was purchased from TCI. β-Propiolactone(98%) was purchased from J&K. 2,6-Bis(1,1-dimethylethyl)-4-methylphenol(BHT), fluorescein diacetate, and 3-(4,5)-dimethylthiahiazo (-z-y1)3,5-diphenytetrazoliumromide(MTT) were purchased from Aladdin. All cell lines were purchased from the China Center for Typical Culture Collection. 2.2. Synthesis of CBAA Monomer.18 (3-Acryloylamino-propyl)(2-carboxy-ethyl)dimethylammonium (CBAA) was synthesized by reacting 4.62 g of DMAPA with 2.97 g of β-propiolactone in 50 mL of anhydrous acetone at 0 °C for 3 h under nitrogen protection. White precipitate obtained was washed with anhydrous acetone and anhydrous ether, dried in vacuum, and stored at 4 °C. Yield: 70%. 1 H NMR (Bruker 400 MHz, D2O): 6.11(t,1H,CHHCH), 6.05 (t, 1H, CHHCH), 5.62(t, 1H, CHHCH), 3.40 (t, 2H, N−CH2− CH2−COO), 3.20(m, 4H, NH−CH2−CH2−CH2), 2.91(s, 6H, N− (CH3)2), 2.49(t, 2H, CH2−COO), 1.89(t, 2H, NH−CH2−CH2− CH2). 2.3. Synthesis of Different Degrees of Modification of PAMAM by CBAA(CBAA-PAMAM). Different amounts of CBAA (4, 8, 16, 64, and 161 mg) were dissolved in 200 μL of a 0.138 M aqueous solution of sodium chloride and mixed with 8 mg of G5 PAMAM in 210 μL of methanol solution. A 0.05 mg amount of BHT was used as polymerization inhibitor of CBAA. Then, the mixture solution was stirred 48 h at room temperature. The mixture was first dialyzed against 0.138 M aqueous sodium chloride solution and then water with a cellulose dialysis membrane (MWCO = 14 000). 2.4. Stability in Protein. Sizes of the native and modified G5 PAMAMs were measured using a Zetasizer Nano ZS system (Malvern, U.K.) equipped with a standard 633 nm laser at a scattering angle of 173°, and the temperature was 37 °C. Measurements were performed in disposable sizing cuvettes. The solution was first filtrated with 100 nm filter needles, each measurement was performed in triplicate, and results were processed with Dispersion Technology Software version 6.01. Each component of the dendrimers of size in PBS was measured. The concentration of native and modified G5 PAMAMs was 1.6 mg/ mL. The Fg solution was first filtrated with 100 nm filter needles. The concentration of Fg was 1 mg/mL. 2.5. Hemolysis Assay. The hemolysis assay was performed according to previous references.22−24 Fresh blood was collected from healthy volunteers in sterile lithium heparin vacutainers. Red blood cells (RBCs) were separated by centrifugation of whole blood diluted in phosphate-buffered saline (PBS) at 1500 rpm for 10 min. RBCs were further washed three times with sterile PBS solution. After the supernatant was removed following the last wash, the cells were resuspended in PBS to get a 2% w/v RBCs suspension. Native and modified G5 PAMAMs were also prepared in sterile PBS. A 150 μL amount of the dendrimers prepared in PBS were added to 150 μL of the 2% w/v RBCs suspension to make the final dendrimers concentration of 10 mg/mL and incubated for 4 h at 37 °C. After incubation, the mixture was centrifuged and the supernatants were transferred to a new 96-well plate. Release of hemoglobin was determined by spectrophotometric analysis of the supernatant at 575 nm. Complete hemolysis was attained using water as the positive

2.6. Cell Membrane Integrity Observation. HUVEC cells were seeded in 96-well tissue culture plates at a density of 10 000 cells/well and cultured in a RPMI 1640 medium with 10% FBS. After cells were incubated 17 h, they were cultured in 100 μL of RPMI 1640 medium with native and modified G5 PAMAMs at a concentration of 2 mg/mL at 37 °C for 24 h. Fluorescein diacetate (FDA) was dissolved in acetone to get 5 mg/mL stock solution. The working solution was freshly prepared by adding 5.0 μL of FDA stock solution to 5.0 mL of PBS. To observe the cell membrane integrity, 200 μL of FDA working solution was added into each well and the cells were incubated for 5 min, the wells were then washed twice with PBS, and the cell morphology was observed using a CCD camera mounted on a Nikon Eclipse Ti series microscope with a 20× lens. Each concentration measurement had three replicate wells. 2.7. Cytotoxicity Assay. Cell viability was assessed using a Vybrant MTT cell proliferation assay kit (Invitrogen, Carlsbad, CA). HUVEC cells were seeded in 96-well tissue culture plates at a density of 10 000 cells/well and cultured in a RPMI 1640 medium with 10% FBS. After cells were incubated 17 h, they were cultured in 100 μL of RPMI 1640 medium with native and modified G5 PAMAMs at various concentrations at 37 °C for 24 h, and then the metabolic activity was determined using a MTT assay. After the medium was removed, 90 μL of RPMI 1640 medium and 10 μL of 12 mM MTT stock solution in PBS were added to each well. Samples were incubated at 37 °C for 4 h. Then, the medium was removed and 100 μL of DMSO was added and incubated for 10 min. The absorbance at 570 nm was read with a 96well plate reader SpectraMaxM2, Molecular Devices, Sunnyvale, CA. Cell viability was expressed as the percentage of absorbance of treated cells with native and modified G5 PAMAMs relative to the absorbance of cells that were not treated. Each measurement had three replicate wells. The cytotoxicity to KB cells was the same procedure except with high-glucose DMEM as cultured medium. 2.8. Atomic Force Microscopy (AFM) Characterization. Samples for AFM were prepared by diluting the G5 PAMAM and CBAA-PAMAM-20 solution with deionized water to a concentration of 10−4 mg/mL. Typically, 10 μL of the final solution was placed directly onto a freshly cleaved mica disk. Droplets on mica were blown away by compressed air after 5 min of adsorption. Samples were dried in air at room temperature. Samples on mica were characterized by tapping mode under ambient conditions using a multimode NanoScope AFM from American VEECO Instruments Inc. Silicon tapping probes having a spring constant of ca. 30 N/m with about 5−10 nm radius were used. 2.9. Statistic Analysis. The hemolysis assay results are presented as mean ± standard deviation (SD) for three repeated samples. Statistical comparisons of differences in the hemolysis results were performed by Student’s t test (Origin 7.0), and p values of less than 0.05 were considered significant.

3. RESULTS AND DISCUSSION 3.1. G5 PAMAM Modification and Characterization. In this study, G5 PAMAM dendrimer was used as the starting material to be modified by different amounts of zwitterionic CBAA (Scheme 1). The peak molecular weight (MW) of G5 PAMAM dendrimer was 26 511 Da measured by MALDI-TOF, slightly smaller than the theoretical MW 28 826. Zwitterionic CBAA with quaternary ammonium cation and carboxyl anion has demonstrated excellent nonfouling property in various protein solutions, serum, and blood plasma.25 The double bond on zwitterionic CBAA can react with the primary amino groups on G5 PAMAM through Michael addition reaction. The molar 8915

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PAMAM. The PEGs or acetyl groups modified PAMAM also show similar positive charge reduction after modification.7,27 AFM provides high-resolution topographic images and other properties at the molecular (or even atomic) level.28 AFM images of the G5 PAMAM (Figure 3a) and CBAA-PAMAM-20 (Figure 3b) show the uniformity of the particle size, suggesting that they are monodispersed particles in aqueous solution. Tomalia et al. systematically studied full generation (from G4 to G10) PAMAM dendrimers by AFM. They demonstrate that the measured diameters are always larger than the heights, indicating that the dendrimer molecules are no longer spherical but instead dome shaped when deposited on a mica surface.29 G5 PAMAM in Figure 3a shows flat structure, while CBAAPAMAM-20 in Figure 3b shows higher feature than the former ones. This indicated that CBAA-PAMAM-20 is more rigid than G5 PAMAM. The size of CBAA-PAMAM-20 is similar to that of the sixth generation of PAMAM. The increase in particle size of CBAA-PAMAM-20 is due to the extra layer of CBAA. 3.2. Nonspecific Interactions between the Modified G5 PAMAM and the Protein. All of the G5 PAMAM and CBAA-PAMAMs were tested for their interactions with Fg by DLS. Fg is a protein found in blood plasma which plays a vital role in blood clotting, which is activated through unfolding of Fg induced by either charge interaction4 or other hydrophobic interaction. Good stability in Fg solution is necessary for in vivo drug delivery or contrast agents. The average diameters of G5 PAMAM and CBAA-PAMAMs are 9.8 ± 2.2 and 12.9 ± 2.4 nm, respectively (Figure 4a). The diameter of CBAA-PAMAMs slightly rises due to the extra layer of CBAA. The diameter of Fg is about 30 nm (Figure 4b), as tested by DLS. The Fg molecule has a collinear, trinodular shape of total length 47.5 nm.30 It is reasonable to show a smaller hydrodynamic diameter in DLS. When G5 PAMAM and CBAA-PAMAM-0.5, CBAAPAMAM-1, CBAA-PAMAM-2, and CBAA-PAMAM-8 incubated with Fg, it is immediately observed large particles (Figure 4c) caused by the conformation change of Fg due to PAMAM

Scheme 1. Surface Primary Amines of G5 PAMAM Dendrimer Were Modified by Different Amounts of CBAAa

a

M, the number of CBAA per dendrimer; 128-M, the number of left primary amines per dendrimer.

ratios of CBAA to primary amine of G5 PAMAM from 0.5:1 to 20:1 in modification solution were used to control the modification degrees. It was reported that there are 5 broad peaks in 1H NMR of G5 PAMAM26 as shown in Figure 1. The new peaks at 2.92 ppm represent the protons at −CH2N+(CH3)2CH2− of CBAA, and the peak at 1.86 ppm represents the protons at the −NHCH2CH2CH2N+− (indicated by bold H) of CBAA. The proton signal at 1.86 ppm is easier to be distinguished than the proton signals at 2.92 ppm. Thus, the 1.86 ppm peak is used to quantify the modification degrees of CBAA on G5 PAMAM. The ratio of the peak at 1.86 ppm to all other protons is used to determine the modification degrees of the Michael addition reaction. When the ratio of CBAA to primary amine of PAMAM was 20:1, the G5 PAMAM was fully modified as shown in Table 1. The zeta potentials of dendrimer and modified dendrimers were measured in PBS (Figure 2). A gradual decrease in positive charge is observed. The zeta potential of G5 PAMAM is about 20 mV, while the zeta potential of the fully modified CBAA-PAMAM-20 is decreased to 2 mV in PBS due to the increase of CBAA modification degree. This consistence with the increase of CBAA modification degree indicates that the CBAA layer can screen the internal positive charge of G5

Figure 1. 1H NMR spectra of G5 PAMAM and the modified PAMAMs by CBAA at different degrees. Ratio of the peak at 1.86 ppm, representing the protons at the −NHCH2CH2CH2 N+− (indicated by bold H) of CBAA, to all other protons is used to determine the modification degrees of the Michael addition reaction. 8916

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Table 1. Parameters of Different Degrees of Modification of PAMAM by CBAA molar ratio of CBAA to primary amine of G5 PAMAM

ratio of characteristic area to other area

number of CBAA conjugated per G5 PAMAM molecule

theoretical primary amine of G5 PAMAM

percentage of surface modification by CBAA

yield

0.5:1 1:1 2:1 8:1 20:1

1:75.65 1:31.77 1:24.11 1:20.49 1:16.01

15 44 67 88 144

128 128 128 128 128

12% 34% 52% 69% 113%*

92% 95% 96% 97% 96%

*

Partial primary amine could be modified by two CBAA molecules to form tertiary amine before the surface of CBAA-PAMAM become overcrowded.

Figure 2. Zeta potentials of G5 PAMAM and the CBAA-modified PAMAM in PBS. Zeta potential decreases gradually from 20 (G5 PAMAM) to 2 mV(CBAA-PAMAM-20) with the increase of CBAA modification degree.

Figure 4. Comparison of diameters of (a) CBAA-PAMAM-20 (1.6 mg/mL), (b) Fg (1 mg/mL), (c) mixture of G5 PAMAM (1.6 mg/ mL) and Fg (1 mg/mL), and (d) mixture of CBAA-PAMAM-20 (1.6 mg/mL) and Fg (1 mg/mL) in PBS solution. Diameter of G5 PAMAM and the partially modified G5 PAMAMs in PBS is similar to CBAA-PAMAM-20. Partially modified G5 PAMAMs could cause aggregation of Fg as observed in c. This indicated that the full CBAA layer on G5 PAMAM could dramatically reduce the interaction force with Fg.

Figure 3. Tapping mode AFM images of G5 PAMAM and CBAAPAMAM-20 on mica show the monodispersion and difference in height. Large size is caused by double-tip effect, which will not have a dramatic effect on height. Image size is 500 nm × 500 nm.

enough to become a coexisting solution, although Fg could be easily denatured to form aggregation. 3.3. Interactions between the Modified G5 PAMAM and Cell Membrane. The interactions between the modified G5 PAMAM and the cell membrane were investigated by hemolysis assay, which focus on the interaction of static cell membrane with the modified G5 PAMAMs in relatively short time. Also, the membrane integrity analysis of the HUVEC cells through long-term incubation was also investigated, which

adsorption. White precipitates can be seen by the bare eye after several minutes. However, there is no large particle detected by DLS when CBAA-PAMAM-20, a complete modification of primary amine by CBAA molecules, incubated with Fg (Figure 4d). The solution can keep clear for days. This indicated that the interaction between Fg and CBAA-PAMAM-20 is weak 8917

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Figure 5. Hemolysis assay of G5 PAMAM and the CBAA-modified G5 PAMAM at different degrees. Results indicated that a full zwitterionic CBAA on G5 PAMAM could dramatically reduce hemolysis of RBCs. Concentration of each dendrimer component is 10 mg/mL. Error bars represent mean ± SD (n = 3), asterisk indicates significant difference (*) p < 0.05; ampersand indicates without significant difference (&) p > 0.05.

Figure 6. Cell morphologies of the HUVEC cells after being cultured 24 h with G5 PAMAM and the modified G5 PAMAMs at a concentration of 2 mg/mL: (a) phase contrast micrographs, (b) fluorescence micrographs. Results indicate that cell membranes were intact in the CBAA fully modified G5 PAMAM solution.

particles with blood components in vitro.23 Thus, the hemolytic activity of the PAMAM and CBAA-PAMAMs on human RBCs were characterized as an initial step to evaluate ex vivo blood biocompatibility.22−24 Figure 5 shows that when H2O and PBS were taken as a positive and a negative control, G5 PAMAM showed serious hemolytic activity. With the increase of the modification degrees, the hemolytic activity of CBAA-PAMAM-

focuses on the interaction of growing cell membrane with the modified G5 PAMAMs. Red blood cell lysis is a widely used method to study polymer−membrane interaction. It gives a quantitative measure of hemoglobin release. Hemolysis in vivo can lead to a series of pathological conditions. Thus, it is a necessary step to evaluate the biocompatibility of intravenously administered nano8918

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Figure 7. Cell viabilities of the HUVEC cells (a) and KB cells (b) after being cultured 24 h with different concentrations of G5 PAMAM and the CBAA-modified G5 PAMAMs, which indicates that a full zwitterionic CBAA layer on G5 PAMAM could reduce the cytotoxicity dramatically. Results are means ± standard deviation (SD) (n = 3).

3.4. Cytotoxicity of the Modified G5 PAMAMs. As a result of the disruption of the cell membrane through hole formation, the cytotoxicity of G5 PAMAM, CBAA-PAMAM-1, CBAA-PAMAM-2, and CBAA-PAMAM-8 showed a similar trend within 2 mg/mL concentration. Cell viabilities of the HUVEC cells (Figure 7a) and the KB cells (Figure 7b) cultured with of PAMAM, CBAA-PAMAM-1, CBAA-PAMAM-2, and CBAA-PAMAM-8 decrease with increasing concentration. All cell viabilities decrease to less than 20% when their concentration reaches 2 mg/mL. However, CBAA-PAMAM20 exhibited negligible cytotoxicity when at concentrations up to 2 mg/mL. This is due to the low interaction force between CBAA-PAMAM-20 and cell membranes, even when cells are growing. It is believed that CBAA-PAMAM-20 is hardly entering the inside of the cells, which is similar to the observation of zwitterionic-coated 16 nm gold nanoparticles.36 The zwitterionic self-assembled monolayers on gold nanoparticles could effectively minimize the nonspecific interactions with either nonphagocytic HUVEC and HepG2 cells or phagocytic RAW 264.7 cells and also the corresponding cell uptake.36 In fact, PAMAM and CBAA-PAMAM-1, CBAA-PAMAM-2, CBAA-PAMAM-8, and CBAA-PAMAM-20 show a gradual decrease in positive charge in PBS solution (Figure 2) since the Michael addition reaction did not dramatically decrease the number of groups which could be protonated at pH 7.4. Most primary amines were changed to secondary amines. Even the fully modified one also has a very large amount of secondary amines. This indicates the surface of CBAA groups screen the positive charge of G5 PAMAM. In the author’s view, three effectors might play key roles in the interactions between the modified PAMAM with protein or cell membranes. First, the lower zeta potential of CBAA-PAMAM-20 compared to others contributes to a decrease in the interactions between CBAAPAMAM-20 and protein or cell membranes. Second, the zwitterionic carboxybetaine on PAMAM could reduce the interaction through forming a hydration layer. The compact thin layer of zwitterionic carboxybetaine on the CBAAPAMAM-20 could overcome the charge-driven adsorption and make the CBAA-PAMAM-20 hardly enter the inside of the cells. Third, the decreased shape change of the fully modified G5 PAMAM could reduce the damage of cell membranes through minimizing the contact between the hydrophobic core of the modified G5 PAMAM and the cell membrane. Thus, all partially modified PAMAM show certain cytotoxicity and only

1, CBAA-PAMAM-2, and CBAA-PAMAM-8 reduced gradually. Moreover, when the G5 PAMAM was fully modified by carboxybetaine groups, the hemolytic activity of CBAAPAMAM-20 decreased to an undetectable level. This phenomenon agrees with the Fg aggregation experiment. The modification level is the key factor to reduce the interaction force between the modified G5 PAMAM and biomacromolecules. The excellent compatibility with RBCs indicates that the CBAA-PAMAM-20 could be a promising candidate for those applications in vivo. The membrane integrity and morphological changes of HUVEC cells treated with G5 PAMAM and the modified PAMAMs were investigated. The morphologies of HUVEC cells after 24 h incubation with G5 PAMAM and CBAAPAMAM-1, CBAA-PAMAM-2, CBAA-PAMAM-8, and CBAAPAMAM-20 at a concentration of 2 mg/mL were investigated. Fluorescence micrographs of the cells stained with FDA and corresponding phase contrast micrographs of HUVEC cells are shown in Figure 6. FDA is hydrolyzed in the cytoplasm by intracellular esterases, and the resulting hydrophilic fluorescein is unable to leave intact cells. Thus, accumulation to high levels indicates the presence of both intracellular enzymatic activity and membrane integrity31,32and hence, cell viability.33 The comparison between fluorescence micrographs of the cells stained with FDA and the corresponding phase contrast micrographs can provide a valid index on the growing states of the cells.34 It can be seen from Figure 6 that the cells grew very well in either pure culture media or media containing CBAA-PAMAM-20. This means that the cytotoxicity of the CBAA-PAMAM-20 was very low and negligible. HUVEC cells with CBAA-PAMAM-20 showed similar fluorescence to the control experiment, indicating that most of polar fluorescein trapped within cells and the membranes of the viable cells were intact. However, HUVEC cells with G5 PAMAM, CBAAPAMAM-1, CBAA-PAMAM-2, and CBAA-PAMAM-8 showed no fluorescence, indicating cytotoxicity was obvious and almost no living cells could be found. Since amine-terminated PAMAM dendrimers can induce formation of holes in cell membranes and the holes generated in the cell membranes allow diffusion of polymers, enzymes, and molecules into and out of the cell,35 these results indicated that all partially modified G5 PAMAM by CBAA may still have similar capability to disrupt cell membranes. This also agrees with the hemolysis assay result. The human red blood cell membranes can keep integrity only exposed to the fully modified G5 PAMAM. 8919

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(amidoamine) dendrimers for targeted cancer therapy. Biomaterials 2011, 32, 3322−3329. (10) Jevprasesphant, R.; Penny, J.; Jalal, R.; Attwood, D.; McKeown, N. B.; D’Emanuele, A. The influence of surface modification on the cytotoxicity of PAMAM dendrimers. Int. J. Pharm. 2003, 252, 263− 266. (11) Ciolkowski, M.; Petersen, J. F.; Ficker, M.; Janaszewska, A.; Christensen, J. B.; Klajnert, B.; Bryszewska, M. Surface modification of PAMAM dendrimer improves its biocompatibility. Nanomedicine: NBM 2012, 8, 815−817. (12) Matsuno, R.; Ishihara, K. Integrated functional nanocolloids covered with artificial cell membranes for biomedical applications. Nano Today 2011, 6, 61−74. (13) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Adsorption of fibrinogen and lysozyme on silicon grafted with poly(2-methacryloyloxyethyl phosphorylcholine) via surface-initiated atom transfer radical polymerization. Langmuir 2005, 21, 5980−5987. (14) Yang, W.; Chen, S. F.; Cheng, G.; Vaisocherova, H.; Xue, H.; Li, W.; Zhang, J. L.; Jiang, S. Y. Film thickness dependence of protein adsorption from blood serum and plasma onto poly(sulfobetaine)grafted surfaces. Langmuir 2008, 24, 9211−9214. (15) Carr, L.; Cheng, G.; Xue, H.; Jiang, S. Y. Engineering the polymer backbone to strengthen nonfouling sulfobetaine hydrogels. Langmuir 2010, 26, 14793−14798. (16) Chen, S. F.; Li, L. Y.; Zhao, C.; Zheng, J. Surface hydration: principles and applications toward low-fouling/nonfouling biomaterials. Polymer 2010, 51, 5283−5293. (17) Zhang, X. a.; Lin, W.; Chen, S.; Xu, H.; Gu, H. Development of a stable dual functional coating with low non-specific protein adsorption and high sensitivity for new superparamagnetic nanospheres. Langmuir 2011, 27, 13669−13674. (18) Yang, W.; Xue, H.; Li, W.; Zhang, J. L.; Jiang, S. Y. Pursuing “zero” protein adsorption of poly(carboxybetaine) from undiluted blood serum and plasma. Langmuir 2009, 25, 11911−11916. (19) Yang, W.; Zhang, L.; Wang, S.; White, A. D.; Jiang, S. Y. Functionalizable and ultra stable nanoparticles coated with zwitterionic poly(carboxybetaine) in undiluted blood serum. Biomaterials 2009, 30, 5617−5621. (20) Chen, S. F.; Cao, Z. Q.; Jiang, S. Y. Ultra-low fouling peptide surfaces derived from natural amino acids. Biomaterials 2009, 30, 5892−5896. (21) Chen, S. F.; Zheng, J.; Li, L. Y.; Jiang, S. Y. Strong resistance of phosphorylcholine self-assembled monolayers to protein adsorption: Insights into nonfouling properties of zwitterionic materials. J. Am. Chem. Soc. 2005, 127, 14473−14478. (22) Chen, H.-T.; Neerman, M. F.; Parrish, A. R.; Simanek, E. E. Cytotoxicity, hemolysis, and acute in vivo toxicity of dendrimers based on melamine, candidate vehicles for drug delivery. J. Am. Chem. Soc. 2004, 126, 10044−10048. (23) Dobrovolskaia, M. A.; Clogston, J. D.; Neun, B. W.; Hall, J. B.; Patri, A. K.; McNeil, S. E. Method for analysis of nanoparticle hemolytic properties in vitro. Nano Lett. 2008, 8, 2180−2187. (24) Yu, T.; Malugin, A.; Ghandehari, H. Impact of silica nanoparticle design on cellular toxicity and hemolytic activity. ACS Nano 2011, 5, 5717−5728. (25) Jiang, S. Y.; Cao, Z. Q. Ultralow-fouling, functionalizable, and hydrolyzable zwitterionic materials and their derivatives for biological applications. Adv. Mater. 2010, 22, 920−932. (26) Majoros, I. J.; Keszler, B.; Woehler, S.; Bull, T.; Baker, J. R. Acetylation of poly(amidoamine) dendrimers. Macromolecules 2003, 36, 5526−5529. (27) Shen, M. W.; Shi, X. Y. Dendrimer-based organic/inorganic hybrid nanoparticles in biomedical applications. Nanoscale 2010, 2, 1596−1610. (28) Bottomley, L. A. Scanning probe microscopy. Anal. Chem. 1998, 70, 425−475. (29) Li, J.; Piehler, L. T.; Qin, D.; Baker, J. R.; Tomalia, D. A.; Meier, D. J. Visualization and characterization of poly(amidoamine)

CBAA-PAMAM-20 crowded by zwitterionic carboxybetaine groups could show very low cytotoxicity.

4. CONCLUSIONS G5 PAMAM dendrimers were surface engineered via the Michael addition reaction between the primary amino group of G5 PAMAM and acrylic functional groups of CBAA. The coverage of CBAA groups can be well controlled from 12% to 113% through adjustment of the molar ratio of CBAA molecules to amino groups of PAMAM dendrimers in modification solution. Hemolysis assay demonstrated that the higher the degree of modification by CBAA, the less human RBCs membrane was disrupted. With full modification, CBAAPAMAM-20 shows undetectable hemolytic activity and very low cytotoxicity, indicating the compact thin layer of zwitterionic carboxybetaine on the CBAA-PAMAM-20 could overcome the charge-driven adsorption. The very low cytotoxicity and excellent biocompatibility of CBAAPAMAM-20 imply the great viability of CBAA-PAMAM-20 for future biomedical applications.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (20974095, 20936005, 21174127) and the Ph.D. Programs Foundation of Ministry of Education of China (20110101110034).



REFERENCES

(1) Svenson, S.; Tomalia, D. A. Dendrimers in biomedical applicationsreflections on the field. Adv. Drug Delivery Rev. 2005, 57, 2106−2129. (2) Liu, J.; Gray, W. D.; Davis, M. E.; Luo, Y. Peptide- and saccharide-conjugated dendrimers for targeted drug delivery: a concise review. Interface Focus 2012, 2, 307−324. (3) Duncan, R.; Izzo, L. Dendrimer biocompatibility and toxicity. Adv. Drug Delivery Rev. 2005, 57, 2215−2237. (4) Jones, C. F.; Campbell, R. A.; Brooks, A. E.; Assemi, S.; Tadjiki, S.; Thiagarajan, G.; Mulcock, C.; Weyrich, A. S.; Brooks, B. D.; Ghandehari, H.; Grainger, D. W. Cationic PAMAM dendrimers aggressively initiate blood clot formation. ACS Nano 2012, 6, 9900− 9910. (5) Jia, L.; Xu, J.-P.; Wang, H.; Ji, J. Polyamidoamine dendrimers surface-engineered with biomimetic phosphorylcholine as potential drug delivery carriers. Colloids Surf., B 2011, 84, 49−54. (6) Wang, W.; Xiong, W.; Wan, J. L.; Sun, X. H.; Xu, H. B.; Yang, X. L. The decrease of PAMAM dendrimer-induced cytotoxicity by PEGylation via attenuation of oxidative stress. Nanotechnology 2009, 20, 105103. (7) Zhu, S. J.; Hong, M. H.; Zhang, L. H.; Tang, G. T.; Jiang, Y. Y.; Pei, Y. Y. PEGylated PAMAM dendrimer-doxorubicin conjugates: in vitro evaluation and in vivo tumor accumulation. Pharm. Res. 2010, 27, 161−174. (8) Majoros, I. J.; Myc, A.; Thomas, T.; Mehta, C. B.; Baker, J. R. PAMAM dendrimer-based multifunctional conjugate for cancer therapy: synthesis, characterization, and functionality. Biomacromolecules 2006, 7, 572−579. (9) Wang, Y.; Guo, R.; Cao, X. Y.; Shen, M. W.; Shi, X. Y. Encapsulation of 2-methoxyestradiol within multifunctional poly8920

dx.doi.org/10.1021/la400623s | Langmuir 2013, 29, 8914−8921

Langmuir

Article

dendrimers by atomic force microscopy. Langmuir 2000, 16, 5613− 5616. (30) Hall, C. E.; Slayter, H. S. The fibrinogen molecule: its size, shape, and mode of polymerization. J. Biophys. Biochem. Cytol. 1959, 5, 11−27. (31) Boender, J. Fluorescein-diacetate, a fluorescent dye compound stain for rapid evaluation of the viability of mammalian oocytes prior to in vitro studies. Vet. Q. 1984, 6, 236−240. (32) Widholm, J. M. The Use of Fluorescein Diacetate and Phenosafranine for Determining Viability of Cultured Plant Cells. Biotech. Histochem. 1972, 47, 189−194. (33) Braschler, T.; Valero, A.; Colella, L.; Pataky, K.; Brugger, J.; Renaud, P. Fluidic microstructuring of alginate hydrogels for the single cell niche. Lab Chip 2010, 10, 2771−2777. (34) Xu, J. P.; Ji, J.; Chen, W. D.; Shen, J. C. Novel biomimetic polymersomes as polymer therapeutics for drug delivery. J. Controlled Release 2005, 107, 502−512. (35) Hong, S.; Bielinska, A. U.; Mecke, A.; Keszler, B.; Beals, J. L.; Shi, X. Y.; Balogh, L.; Orr, B. G.; Baker, J. R.; Banaszak Holl, M. M. Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport. Bioconjugate Chem. 2004, 15, 774−782. (36) Liu, X. S.; Jin, Q.; Ji, Y.; Ji, J. Minimizing nonspecific phagocytic uptake of biocompatible gold nanoparticles with mixed charged zwitterionic surface modification. J. Mater. Chem. 2012, 22, 1916− 1927.

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