Reducing cytotoxicity while improving anti-cancer drug loading capacity of polypropylenimine dendrimers by surface acetylation

Acta Biomaterialia 8 (2012) 4304–4313

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Reducing cytotoxicity while improving anti-cancer drug loading capacity of polypropylenimine dendrimers by surface acetylation Fei Wang a, Xiaopan Cai b,c, Yunzhang Su a, Jingjing Hu d, Q. Wu e, Hongfeng Zhang a,⇑, Jianru Xiao b,⇑, Yiyun Cheng a,⇑ a

Shanghai Key Laboratory of Regulatory Biology and School of Life Sciences, East China Normal University, Shanghai 200241, China Department of Orthopedic Oncology, Changzheng Hospital, The Second Military Medical University, Shanghai 200003, China Department of Orthopedics, The third Affiliated Hospital of Anhui Medical University, Hefeis 230000, China d Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China e Division of Life Sciences, Hongkong University of Science and Technology, Hongkong, China b c

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Article history: Received 6 April 2012 Received in revised form 15 July 2012 Accepted 20 July 2012 Available online 27 July 2012 Keywords: Dendrimer Acetylation PPI Sustained release Anticancer drug

a b s t r a c t Polypropylenimine (PPI) dendrimers have been widely used as effective delivery vehicles for drugs and nucleic acids during the past decade. However, biomedical applications of PPI dendrimers were limited because of their serious cytotoxicity and low drug loading capacity. In the present study, acetylated PPI dendrimers with different degrees of acetylation ranging from 14.2% to 94.3% were synthesized and used to encapsulate drugs, including methotrexate sodium, sodium deoxycholate and doxorubicin. Acetylated PPI dendrimers with a degree of acetylation >80% showed a significantly decreased cytotoxicity (>90% cell viability) on MCF-7 and A549 cells. The drug loading capacity of acetylated PPI dendrimers increased proportionally with the degree of acetylation on the dendrimer surface. In addition, 94.3% acetylated PPI dendrimers exhibited a pH-responsive release profile of anticancer drugs loaded within the nanoparticles. The cytotoxicities of methotrexate sodium and doxorubicin on MCF-7 and A549 cells were significantly reduced when they were complexed with acetylated PPI dendrimers with high degrees of acetylation (>80%), owing to sustained drug release from the dendrimers. The results suggest that surface acetylation can reduce the cytotoxicity and improve the anticancer drug loading capacity of cationic dendrimers, and that acetylated PPI dendrimers are promising vehicles for anticancer drugs in clinical trials. Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Polypropylenimine (PPI) dendrimers are the first commercially available dendrimers synthesized by a divergent strategy using diaminobutane as the central core and propylene imine as repeat units [1]. They are widely used as templates for the synthesis of dendrimer-encapsulated nanoparticles and as scaffolds for the construction of magnetic resonance imaging contrast agents, especially in drug and gene delivery [2–4]. PPI dendrimers have excellent aqueous solubility, and therefore a large number of hydrophobic cavities in their interior can effectively improve the solubility and stability of various hydrophobic drugs [5]. In addition, PPI dendrimers with a high density of active groups on their surface can be easily functionalized with therapeutic agents, targeting moieties, solubilizing ligands and imaging units for targeted cancer diagnosis and therapy [6]. However, PPI dendrimers, especially those with a cationic surface, are not ideal candidates for bio⇑ Corresponding authors. Tel.: +86 021 54342935 (Y. Cheng). E-mail addresses: [email protected] (H. Zhang), [email protected] (J. Xiao), [email protected] (Y. Cheng).

medical applications, owing to their serious toxicity [7–9]. For instance, G5 amine-terminated PPI dendrimer at a low concentration of 1 lg ml1 caused 83.2% and 76.9% cell death on HepG2 and COS-7 cells, respectively [7]. Also, G5 cationic PPI dendrimer showed significant decreases in red blood cell count, hemoglobin content and mean corpuscular hemoglobin value, as well as a substantial increase in white blood cell count [7]. Exposing macrophages to G2 or G3 cationic PPI dendrimers caused dramatic changes in macrophage cell size and significant fluctuation in mitochondrial membrane potential [10]. Cationic PPI dendrimers showed rapid clearance from the blood circulation system after intravenous or intraperitoneal injection, leading to low bioavailability of the administered drugs [11]. Administration of G4 cationic PPI dendrimer caused obvious changes in the behavior of animals, such as decreased food and water consumption, and lower rate of gain in body weight [12]. Besides the non-negligible in vitro and in vivo toxicity, PPI dendrimers have extremely low drug loading capacity for a list of hydrophobic drugs [13]. PPI dendrimers have smaller molecular size and interior cavities compared with polyamidoamine (PAMAM) dendrimers [14]. Though PPI dendrimer with a more hydro-

1742-7061/$ - see front matter Ó 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2012.07.031

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Scheme 1. (a) Synthetic route of acetylated G4 PPI dendrimer, (b) repeated unit of acetylated PPI dendrimer with proton labeling, and molecular structure of (c) methotrexate sodium and (d) doxorubicin.

phobic interior more easily encapsulate hydrophobic compounds, the yielding PPI/drug complexes are not as stable as PAMAM/drug complexes in water and may precipitate from the aqueous solution when PPI dendrimers are loaded with a large number of drug molecules [13]. These defects of PPI dendrimers prevent the development of PPI dendrimer-based drug formulations and diagnostic agents for biomedical purpose [9]. To decrease the cytotoxicity and improve the drug loading capacity of dendrimers, surface engineering of the dendrimer surface by PEGylation [15–18], acetylation [19–21], glycosylation [9] and amino acid or peptide modification [9] was proposed by several groups. Among these strategies, PEGylation and acetylation were considered to be the most efficient ones in reducing dendrimer cytotoxicity and improving their aqueous stability in physiological conditions [22]. Compared with PEGylation, acetylation is preferred for the following reasons: (1) acetylation is more facile and highly efficient, and the degree of acetylation on the dendrimer surface can be easily tailored by choosing proper stoichiometry of acetic anhydride and dendrimer [23,24]; (2) modification of PEG chains with a larger molecular size than acetyl groups on the dendrimer surface will cause significant steric hindrance and thereby affect other functional groups such as targeting moieties

[25]; and (3) acetylated dendrimers can maintain the high penetration ability of cationic dendrimers across cell membranes, while PEGylated dendrimers show much reduced cellular uptake [26]. Generally, acetylation can effectively increase the aqueous solubility of dendrimer–drug conjugates, improve their biocompatibility, and optimize their in vivo pharmacokinetic behavior [9]. Though acetylated dendrimers showed several promising advantages in previous studies, the following questions are still unknown: (1) What is the least degree of acetylation on dendrimer surface that can meet the need of biomedical applications such as drug delivery, gene delivery and disease diagnosis? (2) How will the degree of acetylation influence the drug loading capacity of acetylated dendrimers? (3) Can the acetylated dendrimers with high degrees of acetylation be used directly as drug vehicles for anticancer drugs? The present study addressed these questions using methotrexate sodium, sodium deoxycholate and doxorubicin as model drugs. Sodium deoxycholate was used because it is a widely used amphiphilic guest for dendrimers. G4 PPI dendrimers with different degrees of acetylation ranging from 14.2% to 94.3% were synthesized, characterized by nuclear magnetic resonance (NMR) techniques, and used to encapsulate three model drugs. The drug loading capacity and cytotoxicity of these acetylated

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Fig. 1. 1H NMR spectra of acetylated PPI dendrimers with different degrees of acetylation.

PPI dendrimers were investigated. Also, the biological activities of the acetylated PPI/anticancer drug complexes were evaluated on several cell lines. 2. Experimental section 2.1. Materials G4 diaminobutane-cored PPI dendrimer was purchased from Sigma-Aldrich (St. Louis, MO). Sodium deoxycholate and methotrexate sodium were purchased from Shanghai BBI Co. Ltd (Shanghai, China). Doxorubicin hydrochloride was purchased from Aladdin Chemistry Co. Ltd (Shanghai, China). Acetic anhydride and triethylamine was purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Deuterium oxide (D2O) was obtained from Beijing Chongxi High-Tech Incubator Co. Ltd (Beijing, China). All the chemicals were used as received without further purification. 2.2. Acetylation of PPI dendrimers [24] To prepare acetylated PPI dendrimers with various degrees of acetylation, the molar ratios of acetic anhydride to amine groups on G4 PPI dendrimer were set at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% and 500%. Briefly, different amounts of acetic anhydride were added slowly to a G4 PPI dendrimer (40 mg, 11.3 lmol) solution in methanol (4 ml). Triethylamine (1.2 M equivalent of acetic anhydride) was added to neutralize the yielding acetic acid during the acetylation process (Scheme 1a). The reaction mixtures were stirred at room temperature for 24 h to ensure completion of the acetylation. After that, the solutions were dialyzed extensively against deionized water and followed by PBS buffer (pH 7.4) to remove the acetic ions bound to dendrimers via ionic interactions, and finally the products were again dialyzed against deionized water to remove the salts in the products. The samples were lyophilized to obtain white powders and stored in a dry place for further use. 2.3. 1H NMR analysis The 1H NMR spectra of the acetylated PPI dendrimers with different degrees of acetylation were obtained on a Varian

699.804 MHz NMR spectrometer at 298.2 ± 0.1 K. The 1H NMR spectra of complexes formed between acetylated PPI dendrimers and sodium deoxycholate in D2O were obtained on a Bruker Advance 500.132 MHz NMR spectrometer at 298.2 ± 0.2 K. A certain amount of ethanol was added into the NMR samples as an internal standard, and the complex solutions were sonicated for 2 h before being transferred to NMR tubes. 2.4. 1H–1H two-dimensional nuclear Overhauser effect spectroscopy (NOESY) analysis of the dendrimer–drug complexes The 1H–1H NOESY spectrum of the complex of 94.3% acetylated G4 PPI dendrimer and methotrexate sodium (2 mg acetylated G4 PPI dendrimer and 2.27 mg methotrexate sodium in 500 ll D2O) was obtained on a Varian 699.804 MHz instrument using standard pulse sequences at 298.2 ± 0.1 K. Generally, the 1H–1H NOESY experiments were performed with a 1 s relaxation delay, 97.5 ms acquisition time, and a 7.7 ls 90° pulse width. The mixing time for the optimization of cross-peak intensities was chosen as 300 ms, with minimum distortions during the period for NOE establishment. Sixteen transients were averaged for 1024 complex t1 points. All the data were processed with NMRpipe software on a Linux workstation with standard Lorents–Gauss window function and zero-filling in both dimensions. The 1H–1H NOESY spectrum of the complex consisting of 94.3% acetylated G4 PPI dendrimer and sodium deoxycholate was performed on a Bruker 500.132 MHz instrument using a similar pulse sequence. 2.5. Complex preparation and drug loading capacity Acetylated G4 PPI dendrimers were dissolved in aqueous solutions at a concentration of 4 mM and used as stock solutions. Sodium deoxycholate and methotrexate sodium were dissolved in D2O at concentrations of 24.1 and 20.1 mM, respectively. The aqueous solutions of acetylated PPI dendrimers (pH = 7.2) and aqueous solutions of the methotrexate were mixed together, followed by sonication of the samples for 2 h. The molar ratio of methotrexate/dendrimer was maintained at 16:1 in the preparation of acetylated PPI dendrimer/methotrexate complexes. For sodium deoxycholate, 76 nmol acetylated G4 PPI dendrimer was mixed with 1.21 lmol sodium deoxycholate in 500 ll D2O, while for methotrexate sodium, 30 nmol acetylated G4 PPI dendrimer was

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Fig. 2. 1H NMR spectra of acetylated PPI dendrimer/sodium deoxycholate complexes.

mixed with 0.48 lmol sodium deoxycholate in 200 ll deionized water. The amount of sodium deoxycholate in the G4 PPI dendrimer solution was monitored by 1H NMR spectra, while the complexes of methotrexate sodium and acetylated PPI dendrimer were centrifuged at 10000 rpm for 5 min to remove the precipitates, and the amount of methotrexate sodium in the supernatant was measured by a high-performance liquid chromatography (HPLC) method. The preparation of acetylated G4 PPI dendrimer/ doxorubicin complexes was conducted according to the method adopted in the preparation of sodium deoxycholate and methotrexate sodium complexes, using different amounts of acetylated dendrimers and doxorubicin.

2.6. HPLC analysis The HPLC experiments were conducted on a reverse HPLC instrument (Agilent1200, USA) equipped with a C18 column (4.6 mm in diameter, 150 mm long, 5 lm particle size, ZORBAX Eclipse XDB, Agilent, USA). The mobile phase was methanol and 2% acetic acid at a volume ratio of 20:80 and a flow rate of 1.0 ml min1. A 10 ll sample was injected, and the drug was detected at 313 nm. The retention time of methotrexate sodium was 7.5 ± 0.2 min. The standard curve for methotrexate sodium is: area = 19,181C  6.3535, C in mg ml1; R2 = 0.9984 and the applicable methotrexate concentration ranges from 104 to 102 mg ml1.

Fig. 3. Drug loading capacities of acetylated PPI dendrimers with different degrees of acetylation using methotrexate sodium as a model drug.

2.7. In vitro release studies The release rate of methotrexate sodium from acetylated G4 PPI dendrimer with a 94.3% degree of acetylation was measured by an equilibrium dialysis method. Acetylated G4 PPI/methotrexate complexes were prepared as described above at a drug/dendrimer molar ratio of 5:1. The complex solutions were transferred into a

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Fig. 4. 1H–1H NOESY spectrum of (a) acetylated PPI dendrimer (degree of acetylation 94.3%)/methotrexate and (b) acetylated PPI dendrimer (degree of acetylation 94.3%)/ doxycholate complexes in D2O at a mixing time of 300 ms.

dialysis bag (Spectrum, America) with a molecular weight cut off of 1000 Da, and immersed into 50 ml aqueous solution. The outer phase of the dialysis bag was stirred during the release experiment. At scheduled intervals, 100 ll of the outer phase solutions was withdrawn and replenished with an equal volume of fresh water. The cumulative amount of methotrexate sodium released out of the dialysis bag was measured by the HPLC method. To investigate the effect of environmental pH condition on the release rate of methotrexate sodium, the pH values of outer phase solutions were adjusted to 4.0, 5.0 and 7.0 by 0.1 M HCl and NaOH. 2.8. Cytotoxicity of acetylated G4 PPI dendrimers and their complexes with anticancer drugs MCF-7 (human breast adenocarcinoma cell line, ATCC) and A549 cells (human lung adenocarcinoma epithelial cell line, ATCC) were incubated at 37 °C in 5% CO2 and Dulbecco’s modified Eagle’s medium (DMEM; Gibco Inc.) supplemented with streptomycin, penicillin sulfate and 10% heat-inactived fetal calf serum (Gibco Inc.). Cytotoxicities of acetylated G4 PPI dendrimers (10–20 lM) with different degrees of acetylation and their complexes with two anticancer drugs methotrexate sodium (10 lM) and doxorubicin (0.2–2.5 lM) on MCF-7 and A549 cells were evaluated by a 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acetylated PPI dendrimer/anticancer drug complexes were sonicated for 2 h after sample preparation. MCF-7 and A549 cells were seeded in 96-well plates 48 h before the cyto-

toxicity experiment and treated with acetylated G4 PPI dendrimers or dendrimer/drug complexes for another 48 h. The cells were then washed with PBS buffer and further incubated with MTT reagent for 3 h, and the formazan crystals yielded were dissolved by DMSO. Absorbances of the DMSO solutions in each well were measured at 570 nm using a microplate reader (MQX200R, BioTeK Inc.). Six repeats were conducted for each sample, and the results are shown as mean ± standard deviation. The data were analyzed by twotailed, unpaired Student’s t-tests. To visualize the uptake of doxorubicin and acetylated PPI/doxorubicin complexes by MCF-7 cells, the cells were incubated with 5 lM doxorubicin or doxorubicin/ dendrimer complexes for 3 h, 6 h and 12 h, and washed with PBS buffer. Then the nuclei of MCF-7 cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI). The stained cells were then imaged by a fluorescence microscope (Leika DMI4000 B, Germany). 3. Results and discussion 3.1. Acetylation of G4 PPI dendrimers As shown in Scheme 1b and Fig. 1, cationic G4 PPI dendrimer has five peaks: HA at 1.77 ppm, HB at 2.52 ppm, HB0 at 2.57 ppm, and HC at 2.88 ppm. The peak for protons HD located at the central core of G4 PPI dendrimer is weak and overlaps with that of HA after acetylation [27]. Acetylation of PPI dendrimer generates a new peak at 1.85 ppm, corresponding to methyl protons in the acetyl

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Scheme 2. Proposed inclusion structures of acetylated G4 PPI dendrimer/drug complexes: (a) methotrexate sodium; (b) sodium deoxycholate.

than the theoretical value at acetyl anhydride/amine molar ratios <50%, probably as a result of ionic attachment of acetic acid on the dendrimer surface during the acetylation process [24] and/or errors during the integration of 1H NMR spectra. 3.2. Drug loading capacities of acetylated PPI dendrimers with different degrees of acetylation

Fig. 5. Viabilities of MCF-7 and A549 cells incubated with 0.02 mM acetylated PPI dendrimer with different degrees of acetylation.

group. In addition, a new peak appears at 3.07 ppm, corresponding to the methylene protons HC adjacent to the added acetyl groups [24]. The intensities of peaks at 1.85 ppm and 3.07 ppm increase proportionally with increasing molar ratios of acetyl anhydride and G4 PPI dendrimer, suggesting an increased degree of acetylation on the PPI surface. The decreased intensity of the peak at 2.88 ppm suggests a decreased amount of cationic amine groups on the dendrimer surface. At the same time, peaks for protons HB and HB0 shift to downfield, owing to the decreased electron density around these protons after acetylation. The degrees of acetylation of G4 PPI dendrimer were calculated from the integration areas of the peaks corresponding to PPI scaffold protons and the added acetyl groups in 1H NMR spectra. Peaks HA, HB, HB0 and HC of G4 PPI dendrimer represent 368 protons, while the peak at 1.85 ppm corresponds to the methyl protons in the added acetyl groups. By comparing the integrated areas of these peaks, the average number of acetyl groups conjugated to each G4 PPI dendrimer can be calculated, and the degrees of acetylation are shown in Fig. 1 [28–30]. As expected, the acetylated dendrimers are obtained in high yield (>95%), and the calculated degree of acetylation is in direct proportion to the added amount of acetyl anhydride. Surprisingly, it can be found that the calculated degree of acetylation on G4 PPI dendrimer is slightly larger

When sodium deoxycholate was added to solutions of nonmodified PPI dendrimers or acetylated PPI dendrimers with low degrees of acetylation, white precipitates were observed in the complex solutions. As shown in Fig. 2, strong NMR signals of deoxycholate (0.573 ppm, 0.785 ppm and 3.919 ppm) were observed in the 1H NMR spectrum of unmodified PPI/deoxycholate complex because the unmodified PPI dendrimer/deoxycholate precipitate consists of a high percentage of PPI dendrimer and a low percentage of deoxycholate. Convincing evidence is the disappearance of PPI peaks when the degree of acetylation is 0%, while in the presence of higher degree of acetylation PPI dendrimers, e.g., 88.9% and 94.3%, the appearance of strong NMR peaks for deoxycholate is due to the stability of acetylated PPI/deoxycholate complex in aqueous solution, and both the NMR peaks of PPI and deoxycholate are therefore observed in Fig. 2. For acetylated PPI dendrimers with degrees of acetylation 14.3–69.7%, the acetylated PPI dendrimer/ deoxycholate precipitates have a high percentage of deoxycholate and a relatively low percentage of PPI dendrimer. As a result, deoxycholate signals were observed only in the unmodified PPI dendrimer and the almost fully acetylated dendrimer complex solutions. Acetylated PPI dendrimers with degree of acetylation >88.9% can prevent the precipitation of dendrimer/deoxycholate complexes. As demonstrated in previous studies, unmodified G4 PPI dendrimer failed to significantly enhance the aqueous solubility of phenylbutazone [13]. Though NOESY studies revealed the encapsulations of phenylbutazone molecules within the cavities of G4 PPI dendrimer, phenylbutazone loaded within PPI dendrimer above a critical concentration causes the precipitation of the PPI dendrimer/phenylbutazone complexes. This is similar to the phenomenon of PPI dendrimer/deoxycholate complexes in Fig. 2. Similarly, the drug loading capacities of acetylated PPI dendrimers were evaluated using methotrexate sodium (Scheme 1c) as a model drug. The molar ratio of methotrexate/dendrimer is kept

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Fig. 6. Cytotoxicities of 10 lM methotrexate sodium on (a) MCF-7 and (b) A549 cells in the absence and presence of acetylated PPI dendrimers with different degrees of acetylation.

at a constant 16 throughout the drug loading experiment. As shown in Fig. 3, the concentrations of methotrexate sodium in dendrimer/methotrexate complex increase linearly with degree of acetylation of G4 PPI dendrimer until it reaches 88.9%. At higher degrees of acetylation, drug concentrations in the complex solutions are slightly changed, which is in accordance with the results of acetylated PPI dendrimer/deoxycholate complexes. In previous studies, methotrexate was found to form precipitates with cationic PAMAM dendrimers owing to the presence of two carboxyl groups in the methotrexate molecules [31]. Here, the unmodified PPI dendrimer has a nature similar to that of cationic PAMAM dendrimers, and only a low concentration of methotrexate is recovered in the unmodified PPI/methotrexate complex solution. Therefore, unmodified PPI dendrimers are not suitable for the delivery of sodium deoxycholate (Fig. 2) and methotrexate sodium (Fig. 3), owing to the low drug loading capacity of this cationic carrier and the instability of their drug complexes. To confirm the encapsulation of methotrexate sodium and sodium deoxycholate within the interior cavities of acetylated PPI dendrimers, 1H–1H NOESY spectra of 94.3% acetylated dendrimer/methotrexate complex was conducted. The presence of NOE cross-peaks between two protons in the NOESY spectrum indicates spatial proximity of these protons [32–34], The cross-peak intensity decreases with the spatial distance between the protons, and increases with the number of molecules involved in the cross-peak [33]. As shown in Fig. 4a, strong cross-peaks between methotrexate protons (H1, H4–8) and the interior protons of acetylated PPI dendrimer (HA, HB, HB0 , HC) were observed, proving the formation of inclusion structures of methotrexate with acetylated PPI dendrimer. Further in vitro release studies revealed that 5% of the methotrexate in the complex solutions is free drugs, and most of the methotrexate forms stable complexes with acetylated PPI dendrimer. Similarly, cross-peaks between sodium deoxycholate and acetylated PPI dendrimers are observed in the 1H–1H NOESY spectra of acetylated PPI dendrimer (94.3%)/deoxycholate complex (Fig. 4b). These results confirmed that acetylated G4 PPI dendrimers with a degree of acetylation >84.3% can effectively load amphiphilic and hydrophobic drug molecules and form stable inclusion complexes with these drugs (Scheme 2). 3.3. Cytotoxicities of acetylated PPI dendrimers with different degrees of acetylation Fig. 5 shows that the cytotoxicities of acetylated PPI dendrimers depend on their degree of acetylation. The viabilities of cells incu-

Fig. 7. In vitro release rate of methotrexate sodium from acetylated PPI dendrimer (degree of acetylation 94.3%) in water at different pH conditions.

bated with acetylated PPI dendrimers increase gradually with degree of acetylation on both MCF-7 and A549 cells. In the presence of 0.02 mM unmodified G4 PPI dendrimer, the viabilities of MCF-7 and A549 cells are 53.8% and 18.4%, respectively. 14.2– 23.4% acetylation of G4 PPI can slightly decrease cytotoxicity of the unmodified cationic dendrimer, while acetylated PPI dendrimers with higher degrees of acetylation exhibit significantly reduced toxicity on both cells. As the degree of acetylation of dendrimer increases from 33.8% to 40.6%, the viability of MCF-7 cells increases from 64.2% to 89.5%, suggesting that acetylated PPI dendrimer with a degree of acetylation >40.6% is relatively biocompatible on MCF-7 cells at 0.02 mM. Also, it is found that the cytotoxicities of acetylated PPI dendrimers on MCF-7 and A549 cells are scarcely changed above the degree of acetylation of 84.3%. Dendrimer biocompatibility is of central importance in the design of dendrimer-based drug delivery systems [9]. A large number of researchers have reported the in vitro and in vivo toxicity of PPI dendrimers [7,8]. To decrease the toxicity of PPI dendrimer on normal cells and reduce their hemolytic activities on blood cells, acetylation is widely used as an efficient method to solve the issue of the toxicity of cationic dendrimers [22,26]. Acetylation of PPI dendrimers can neutralize their surface cationic charges and decrease their binding affinities with amphiphilic phospholipids as well as biomacromolecules, maintaining the integrity of the cell membrane and preventing the leakage of intracellular components [22].

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Fig. 8. Cytotoxicities of doxorubicin on (a) MCF-7 and (b) A549 cells in the absence and presence of acetylated PPI dendrimers with different degrees of acetylation.

Fig. 9. Images of MCF-7 cells incubated with doxorubicin and acetylated PPI dendrimer/doxorubicin complexes for 12 h; the nuclei of the MCF-7 cells were stained by DAPI.

Here, the effect of degree of acetylation on the cytotoxicity of acetylated PPI dendrimers on MCF-7 and A549 cells was systematically investigated. Cell viabilities of the dendrimers with high de-

grees of acetylation (>84.3%) are >95%. These dendrimers can be considered non-toxic biomaterials in drug delivery. Thus, acetylated PPI dendrimers with degrees of acetylation of 84.3%, 86.4%,

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88.9% and 94.3% were employed in further anticancer drug loading and cytotoxicity evaluation experiments. 3.4. Anticancer drug delivery efficiencies of acetylated PPI dendrimers The obstacle of most anticancer drugs in cancer chemotherapy is their serious side effects. To reduce the side effects of anticancer drugs on healthy tissues, drug delivery systems are proposed as an effective strategy in clinical trials [30]. Anticancer drug molecules conjugated to or encapsulated within a macromolecular carrier can achieve sustained drug release and thus diminish the side effects of anticancer drugs [35]. Recently, dendrimers and dendritic polymers have become hot research topics in this area and have attracted increasing attention from miscellaneous fields [36,37]. Here, four acetylated PPI dendrimers with degrees of acetylation >84.3% were used as biocompatible carriers to deliver anticancer drugs such as methotrexate sodium and doxorubicin to MCF-7 and A549 cells. As shown in Fig. 6a, methotrexate sodium at a concentration of 10 lM killed 50.6% of the MCF-7 cells, while the same amount of methotrexate encapsulated within acetylated PPI dendrimer only killed 19.1–24.5% MCF-7 cells, depending on the degree of acetylation of the PPI dendrimer. Similar results were obtained on A549 cells (Fig. 6b), in which 10 lM free methotrexate showed a cell viability of 34.6%, while the same amount of methotrexate loaded within acetylated PPI dendrimers exhibited cell viabilities of 64.5–70.0%, depending on the degree of acetylation. To confirm the sustained release of methotrexate sodium from acetylated PPI dendrimers, release experiments for anticancer drugs from the complexes at different pH conditions were conducted. As shown in Fig. 7, the release rate of methotrexate from acetylated PPI dendrimer with an acetylation ratio of 94.3% in deionized water is extremely low, and only 5.96% of the drugs released out of the dialysis bag after 12 h. Previous studies have demonstrated that PPI dendrimer have hydrophobic cavities and can encapsulate a number of hydrophobic compounds in its interior [38]. Methotrexate sodium, sodium deoxycholate and doxorubicin molecules in this study are all amphiphilic compounds with hydrophobic regions, and thus can form stable complexes with the acetylated PPI dendrimer. In addition, surface acetylation prevents the ionic adsorption of drugs on the dendrimer surface. Therefore, the release of drug molecules from acetylated PPI dendritic matrix is much slower than that from unmodified PPI dendrimers in previous studies. Fig. 7 also revealed that the release of methotrexate from acetylated PPI dendrimer can be tailored by changing the pH condition of the release system. An acidic environment can stimulate the release of methotrexate molecules. The drug release rate is much faster at pH 4.0. The higher release rate of methotrexate in an acidic environment is probably due to the quaternization of tertiary amine groups in the interior pockets of PPI dendrimer. The quaternization process decreases the hydrophobicity of PPI pockets and increases the dendrimer volume, which accelerate the release of encapsulated anticancer drugs [39]. This pH-responsive release behavior is very important for dendrimer-based drug delivery systems. Dendrimers and dendrimer/drug complexes localize mainly in endosomes after cellular uptake [40]. The pH values of endosomes can drop below pH 5.0 [41], which stimulates the release of encapsulated anticancer drugs. In addition, another anticancer drug doxorubicin (Scheme 1d) was used to evaluate the anticancer drug delivery efficiency of acetylated PPI dendrimers. As shown in Fig. 8a and b, doxorubicin loaded within acetylated PPI dendrimer showed much reduced cytotoxicity on MCF-7 and A549 cells compared with free doxorubicin. It is worth noting that doxorubicin is much more sensitive on A549 cells than on MCF-7 cells. Therefore, different doses of doxorubicin were used on these cells.

To confirm that the anticancer drugs loaded within acetylated PPI dendrimers can be released in cancer cells, MCF-7 cells were incubated with doxorubicin and acetylated PPI dendrimer/doxorubicin complexes and imaged by fluorescence microscopy. As shown in Fig. 9, high efficient uptake of free doxorubicin by MCF-7 cells and co-localization of doxorubicin and the cellular nuclei are observed after 12 h of incubation. In the case of acetylated PPI/doxorubicin complexes, most of the cells are observed with red fluorescence, and a high percentage of the cells show pink nuclei, suggesting the effective delivery of doxorubicin into the nuclei of MCF-7 cells by acetylated PPI dendrimers. These results suggest that acetylated PPI dendrimers with degrees of acetylation of 84.3%, 86.4%, 88.9% and 94.3% are beneficial in the delivery of anticancer drugs such as methotrexate and doxorubicin. 4. Conclusions Acetylated G4 PPI dendrimers with different degrees of acetylation were synthesized and characterized by NMR techniques. The drug loading capacity and cytotoxicity of acetylated PPI dendrimers depend much on the degree of acetylation. Acetylated PPI dendrimers with high degrees of acetylation >84.3% can effectively decrease the dendrimer cytotoxicity and improve their drug loading capacity. Anticancer drugs loaded within acetylated PPI dendrimers exhibited sustained and pH-dependent drug release behavior. Anticancer drugs loaded within the acetylated PPI dendrimers with degrees of acetylation >84.3% can effectively decrease their cytotoxicities on MCF-7 and A549 cells. This study provides new insights into the design of biocompatible dendrimers for anticancer drug delivery. Acetylated PPI dendrimers with high degrees of acetylation are promising drug carriers with low cytotoxicity and high drug loading efficiency. Acknowledgments The authors thank the Talent Program of East China Normal University (No.77202201), the ‘‘Dawn’’ Program of Shanghai Education Commission (No.10SG27), and the Innovation Program of Shanghai Municipal Education Commission (No.12ZZ044) for financial support on this project. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs 1, 2, 4, 7 and 9 and Schemes 1 and 2 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2012.07.031. References [1] de Brabander-van den Berg EMM, Meijer EW. Poly(propylene imine) dendrimers: large-scale synthesis by hetereogeneously catalyzed hydrogenations. Angew Chem 1993;32:1308–11. [2] Gajbhiye V, Jain NK. The treatment of glioblastoma xenografts by surfactant conjugated dendritic nanoconjugates. Biomaterials 2011;32:6213–25. [3] Nir I, Aserin A, Libster D, Garti N. Solubilization of a dendrimer into a microemulsion. J Phys Chem B 2010;114:16723–30. [4] Taratula O et al. Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery. J Control Release 2009;140:284–93. [5] Gupta U, Agashe HB, Asthana A, Jain NK. Dendrimers: novel polymeric nanoarchitectures for solubility enhancement. Biomacromolecules 2006;7:649–58. [6] Kesharwani P, Tekade RK, Gajbhiye V, Jain K, Jain NK. Cancer targeting potential of some ligand-anchored poly(propylene imine) dendrimers: a comparison. Nanomedicine 2011;7:295–304. [7] Jain K, Kesharwani P, Gupta U, Jain NK. Dendrimer toxicity: let’s meet the challenge. Int J Pharm 2010;394:122–42. [8] Duncan R, Izzo L. Dendrimer biocompatibility and toxicity. Adv Drug Deliv Rev 2005;57:2215–37.

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