Enhancement of the Cytotoxicity and Selectivity of Doxorubicin to

Apr 23, 2014 - Department of Physical Medicine and Rehabilitation, National Taiwan ... Cancer has been one of the most common causes of death in...
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Enhancement of the Cytotoxicity and Selectivity of Doxorubicin to Hepatoma Cells by Synergistic Combination of Galactose-Decorated γ‑Poly(glutamic acid) Nanoparticles and Low-Intensity Ultrasound Wei-Bor Tsai,*,† Hsin-Yu Lai,† Jyun-Lin Lee,‡ Chia-Wen Lo,‡ and Wen-Shiang Chen*,‡ †

Department of Chemical Engineering, National Taiwan University, Number 1, Section 4, Roosevelt Road, Taipei 106, Taiwan Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei 100, Taiwan

‡

ABSTRACT: Specific drug delivery to solid tumors remains one of the challenges in cancer therapy. The aim of this study was to combine three drug-targeting strategies, polymer-drug conjugate, ligand presentation and ultrasound treatment, to enhance the efficacy and selectivity of doxorubicin (DXR) to hepatoma cells. The conjugation of DXR to γ-poly(glutamic acids) (γ-PGA) decreased the cytotoxicity of DXR, while the conjugation of galactosamine (Gal) to the γ-PGA−DXR conjugate restored the cytotoxic efficacy of DXR on hepatoma cells due to increased uptake of DXR. Furthermore, lowintensity ultrasound treatment increased the cell-killing ability of γ-PGA−DXR conjugates by 20%. The in vitro results showed the potential of the γ-PGA−DXR−Gal conjugate for future clinical applications.

1. INTRODUCTION

Another targeting strategy, molecular target therapy, focuses on development of ligand-conjugated drug carriers that recognize specific cell membrane markers or receptors that are overexpressed in cancerous cells or tissues.5,6 For example, monoclonal antibody-conjugated anticancer drugs that recognize specific cancerous cells have been applied to treat colorectal cancer clinically.7 Another example is that cytotoxic efficacy of anticancer drugs in hepatoma tissues can be improved by conjugation of the drugs with ligands of asialoglycoprotein receptors (ASGPR),8 that are overexpressed on hepatoma cells.9 The targeting ability of a polymer−DXR conjugate bearing galactosamine to the liver tumor has been demonstrated in a phase I study.10 Physical stimulants such as ultrasound (US) and electricity can facilitate drug delivery to a specific tissue.11,12 US treatment has been shown to enhance cytotoxic efficacy of anticancer agents such as doxorubicin (DXR), cisplatin and 5-fluorouracil.13−15 Cochran et al. showed that US administration enhanced delivery of DXR-loaded microbubbles to hepatoma tissues in a rat model, and effectively reduced growth of the tumor tissues with lower levels of DXR in the plasma and the myocardium.16 The aim of this study is to combine the above three drugtargeting strategies for enhancing the efficacy and selectivity of anticancer drugs. γ-Poly(glutamic acids) (γ-PGA), a natural poly(amino acid) synthesized by certain strains of Bacillus,8 was

Cancer has been one of the most common causes of death in developed countries, and the incidence of cancer keeps elevating worldwide. Systemic chemotherapy is a traditional therapeutic approach for the treatment of localized and metastasized cancers. However, delivery of anticancer drugs to solid tumors remains a challenge in chemotherapy. A major drawback in systemic chemotherapy is that most of the anticancer drugs lack tumor selectivity, so normal tissues are also damaged by the anticancer agents, leading to systemic toxicity that limits the usage of anticancer drugs.1 Thus, development of drug delivery systems for specific targeting to tumor tissues has been the focus of many studies in order to improve the therapeutic efficacy and to decrease the systemic toxicity of anticancer drugs. Several drug-targeting strategies have been developed for cancer therapies. Passive targeting that is based on the enhanced permeation and retention (EPR) effect,2 uses drug nanocarriers that spontaneously accumulate at tumor sites, due to enhanced vascular permeability of tumor tissue, compared to normal tissues. This characteristic could facilitate leakage of macromolecules larger than 40 kDa from vascular walls to tumor tissues so that accumulation of large macromolecules in tumor tissues is increased.3 Polymer-drug conjugates take the advantage of the enhanced EPR effect of increased residence time in circulation. Several polymer-drug conjugates have demonstrated merits compared to the corresponding parent drugs, such as increased therapeutic efficacy and fewer side effects, and have advanced to the clinical trial stage.4 © 2014 American Chemical Society

Received: January 25, 2014 Revised: April 18, 2014 Published: April 23, 2014 5510

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Table 1. Compositions of Reaction Mixtures for the Conjugation of DXR and Gal to γ-PGA with Molecular Weight of 90 or 1200 kDaa γ-PGA reaction ratio 1/0 1/1 1/4 2/0 2/2 2/8 a

90

1200 1.00 1.00 1.00 1.00 1.00 1.00

EDC

NHS

DXR

Gal

90

1200

90

1200

90

1200

90

1200

1.40 1.40 1.40 1.40 1.40 1.40

1.48 1.48 1.48 1.48 1.48 1.48

0.84 0.84 0.84 0.84 0.84 0.84

0.89 0.89 0.89 0.89 0.89 0.89

0.40 0.40 0.40 0.80 0.80 0.80

0.42 0.42 0.42 0.84 0.84 0.84

0 0.16 0.63 0 0.31 1.26

0 0.17 0.67 0 0.34 1.33

The units for the values are in mg/mL.

used as a drug carrier. γ-PGA is water-soluble, biodegradable, and has low toxicity. γ-PGA-based amphiphilic polymers has been recently used to fabricate self-assembled nanoparticles for encapsulation of anticancer drugs.8,17 In this study, γ-PGA was used directly as a drug carrier. An advantage of γ-PGA is that it contains abundant carboxyl groups for conjugation of drugs and ligands. A model drug, doxorubicin (DXR), and an ASGPR ligand, galactosamine, were conjugated to γ-PGA simultaneously via carbodiimide chemistry. The cytotoxicity and selectivity of γPGA-derived drug nanocarriers were then evaluated in vitro. Finally, US was applied to enhance the cell-killing efficacy of γPGA−DXR conjugates.

their derivatives were determined by Acoustic and Electroacoustic Spectrometer (Malvern, Nanosizer, USA). DXR conjugation was quantified by UV absorbance at 480 nm.19 Gal conjugation was determined by the Elson-Morgan assay.20 Briefly, Gal-grafted γ-PGA was dissolved in 4 M HCl and then flushed with dry nitrogen. After incubation in a 100 °C oil bath for 4 h, the hydrolysate was dried in a vacuum oven. The samples were dissolved in p-dimethylaminobenzaldehyde solution, and then heated at 75 °C for 30 min. Finally, the absorbance of the solution at 530 nm was read. 2.4. Cytotoxicity Assay. Cytotoxicity of the drug conjugates was determined by the MTT test.21 Briefly, BNL cells or L929 cells were seeded in 24-well plates at 4 × 104 cells/cm2 overnight. The culture medium was then replaced with fresh culture medium containing 100− 500 ng/mL DXR in the form of free, γ-PGA−DXR or γ-PGA−DXR− Gal. After overnight culture, the cells were incubated in 4 mg/mL 1-(4,5dimethylthiazol-2-yl)-3,5-diphenylformazan solution for 4 h at 37 °C. The formed formazan crystals were dissolved in DMSO, and the content of formazan was determined by the absorbance at 570 nm using an ELISA reader (Model EL800, BIO-TEK, USA). Cell viability was presented as a ratio to the drug-free control. 2.5. Analysis of Cellular Uptake of DXR. DXR, γ-PGA90−DXR, or γ-PGA90−DXR−Gal at various concentrations was added in cell culture media for 0.5 or 3 h of incubation. For observation of cellular uptake of DXR using fluorescent microscopy, the cells were fixed in 4% paraformaldehyde for 10 min. The cells were then permeated with 0.1% Triton X-100 for 5 min. The uptake of DXR was observed using a fluorescent microscope (OLYMPUS, BX51, USA). The cell nuclei were counter-stained with 4′,6′-diamidino-2-phenylindole (DAPI, Invitrogen, USA). For quantification of cellular DXR uptake, after being incubated with DXR, γ-PGA90−DXR(2/0), or γ-PGA90−DXR−Gal(2/8) for 3 h, the cells were lysed by 0.1% Triton X-100 at 4 °C for 15 min. The lysates were centrifuged at 12 000 rpm for 10 min, and the fluorescence intensities of the supernatants were determined by a fluorometer at an excitation wavelength of 460 nm and emission wavelength of 595 nm.22 2.6. Ultrasound Treatment. The US setup was described in our previous report.23 BNL cells at 5 × 104/cm2 were seeded in TCPS 24well plates overnight. The culture medium was replaced with fresh media containing 100−700 ng DXR/mL in the form of free DXR, γPGA90−DXR or γ-PGA90−DXR−Gal. The cell culture was then treated with ultrasound (US) at 1 MHz frequency and 2 W/cm2 for 1 min. Such US treatment was previously proved harmless to cells.24 After 24 h of culture, the MTT assay was used to determine cell viability. 2.7. Statistic Analysis. The data was presented as means ± standard deviation. The statistical differentiations between different samples were determined by Student-Newman- Keuls Multiple Comparisons Test (Instat 3.0, Graph Pad Software, USA). Probability p ≤ 0.05 was considered as the existence of a significant difference between different groups.

2. MATERIALS AND METHODS 2.1. Materials. Most of the reagents were purchased from Sigma− Aldrich (USA) unless specified otherwise. N-hydroxysuccinimide (NHS), N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC), and galactosamine hydrochloride (Gal, cat #1772-038) were purchased from Sigma-Aldrich. Doxorubicin (DXR, cat# 286054) was purchased from Pfizer, Italy. Gamma-poly(glutamic acids) (γ-PGA, molecular weight ca. 1200 kDa; abbreviated as γ-PGA1200) was received from VEDAN Enterprise Corporation (Taiwan). The γPGA was further hydrolyzed in a tightly sealed steel container at 150 °C,18 and the hydrolyzed γ-PGA was then fractioned by gel filtration. γPGA with a molecular weight of 90 kDa (γ-PGA90) was collected and used in this study. Mouse L929 fibroblasts and BNL hepatoma cells were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). BNL cell culture medium consisted of Dulbecco’s Modified Eagle medium (HyClone, USA), 10% (v/v) fetal bovine serum (FBS, JRH Biosciences, Australia), 3.7 mg/mL NaHCO3, 0.5% (v/v) fungizone (GIBCO, Invitrogen, USA), 0.25% (v/v) gentamicin (GIBCO), and 1.358% (v/v) β-mercaptoethanol. L929 cell culture medium consisted of α-minimum essential medium (HyClone, USA), 10% (v/v) FBS, 2 mg/mL NaHCO3, 0.5% (v/v) fungizone, 0.25% (v/v) gentamicin, and 0.679% (v/v) β-mercaptoethanol. Phosphate buffered saline (PBS, pH 7.4) contained 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4. 2.2. Conjugation of γ-PGA with Doxorubicin and Galactosamine. γ-PGA was conjugated with DXR and Gal via a carbodiimide reaction. γ-PGA, EDC, NHS, and Gal were dissolved individually in deionized water, while DXR was dissolved in dimethyl sulfoxide (DMSO). γ-PGA solution was mixed with EDC and NHS prior to addition of DXR and/or Gal. A series of γ-PGA derivatives were synthesized according to the molar ratios of DXR and Gal to glutamic acids of γ-PGA (Table 1). For example, the abbreviation of 1/4 in Table 1 represents that the reaction mixture contains one DXR and 4 Gal per 10 glutamic acid units. After reaction, γ-PGA−DXR and γ-PGA−DXR− Gal were purified using gel filtration with Sephedex-G75. 2.3. Characterization of γ-PGA−DXR and γ-PGA−DXR−Gal. The conjugation of DXR and Gal was verified by H1 nuclear magnetic resonance spectroscopy (NMR, Varian UNITY INOVA-500, USA). γPGA−DXR and γ-PGA−DXR−Gal were dissolved in d-DMSO, while γ-PGA was dissolved in D2O. The sizes and zeta-potentials of γ-PGA and

3. RESULTS AND DISCUSSION 3.1. Characterization of γ-PGA−DXR and γ-PGA−DXR− Gal. The H1-NMR spectrum of our γ-PGA (Figure 1) is almost identical to a reported γ-PGA NMR spectrum.25 The appearance 5511

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Figure 1. H1 NMR spectra of (A) γ-PGA, (B) γ-PGA−DXR(2/0), and (C) γ-PGA−DXR−Gal(2/8).

of peaks between 6.5 and 7 ppm (aromatic hydrogen from DXR) and a peak near 2.4 ppm (methyl hydrogen) indicates successful DXR conjugation (Figure 1). The spectrum of γ-PGA−DXR− Gal was similar to that of γ-PGA−DXR (Figure 1), probably because Gal only contains hydroxyl hydrogen, whose NMR

characteristic peak is similar to that of hydroxyl hydrogen in DXR. The amounts of conjugated DXR and Gal in the γ-PGA derivatives are listed in Table 2. DXR conjugation at a ratio of 1 per 10 glutamic acids was around 200 and 250 μg/mg γ-PGA for 5512

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Table 2. Characterization of DXR and Gal Conjugated γ-PGA with Molecular Weight of 90 or 1200 kDaa DXR conjugation (μg/mg)

a

reaction ratio

90

1200

γ-PGA 1/0 1/1 1/4 2/0 2/2 2/8

194 190 182 427 411 340

244 256 194 454 463 396

Gal conjugation (μg/mg) 90

1200

4 94

ND 21

18 293

ND 44

diameter (nm)

zeta potential (mV)

90

1200

90

1200

5−15 103 95 50−120 147 159 10−24

7−25 135 167 60−120 206 204 90−220

−35.0 −26.4 −32.0 −32.9 −27.8 −31.1 −32.0

−37.8 −39.4 −43.1 −43.4 −50.0 −74.6 −68.6

The reation ratio x/y represents x DXR and y Gal for every 10 glutamic acids in the reaction mixture. ND: not detected.

γ-PGA90 and γ-PGA1200, respectively. DXR conjugation was increased to ∼440 μg/mg γ-PGA for both γ-PGA90 and γPGA1200 at a higher conjugation ratio (2 per 10 glutamic acids). Gal conjugation at lower ratios did not affect DXR conjugation, while higher Gal contents (4 or 8 per 10 glutamic acids) decreased 10−20% of DXR conjugation to γ-PGA. Since the charges of nanoparticles may affect their interactions with cells, the zeta potentials of γ-PGA and its derivatives were analyzed (Table 2). The zeta potential of γ-PGA was ∼ −37 mV, regardless of molecular weights. DXR conjugation slightly increased the zeta potential of γ-PGA90 to ∼ −27 mV, while Gal conjugation slightly decreased the zeta potential (−32 mV). On the other hand, the conjugation of both DXR and/or Gal decreased the zeta potentials of γ-PGA1200 up to −75 mV. In conclusion, γ-PGA90 and their derivatives remained negatively charged within −26 mV to −35 mV, while γ-PGA1200 and their derivatives possessed zeta potentials lower than −37 mV. The diameters of γ-PGA, γ-PGA−DXR and γ-PGA−DXR− Gal were determined since the sizes of nanoparticles affect the efficiency of cell uptake and infiltration into cancer tissues.26−28 γ-PGA90 and γ-PGA1200 were 5−15 nm and 7−25 nm in diameter, respectively (Table 2). The wide size-distribution of γPGA indicates the molecular-weight heterogeneity in our γ-PGA. DXR conjugation significantly increased the size of γ-PGA. The sizes of γ-PGA90−DXR were 103 and 147 nm for γ-PGA90− DXR (1/0) and γ-PGA90−DXR (2/0), respectively. Similarly, γPGA1200−DXR were 135 and 206 nm in diameters for γPGA1200−DXR(1/0) and γ-PGA1200−DXR(2/0), respectively. We conjecture that the significant increase in the sizes of γ-PGA−DXR conjugates is attributed to the hydrophobicity of DXR, which causes aggregation of γ-PGA−DXR in aqueous solutions.29 On the contrary, incorporation of galactosamine increases the hydrophilicity and dissolvability of DXR conjugates, and thus decreases the size of γ-PGA−DXR. For example, the size of γ-PGA90−DXR was decreased from 147 nm (γ-PGA−DXR 2/0) to 10−24 nm (γ-PGA−DXR 2/8) after conjugation of Gal. 3.2. Cytotoxicity of γ-PGA−DXR and γ-PGA−DXR−Gal. The cytotoxicity of γ-PGA was first examined in order to evaluate its safety as a drug carrier. The MTT assay indicated that the cell viability after incubation with 1−10 μg/mL γ-PGA90 and γPGA1200 remained above 90% (Figure 2). Since in the subsequent cell experiments, the contents of γ-PGA in the γPGA−DXR conjugates were all below 5 μg/mL, the cytotoxicity of γ-PGA in the drug conjugates could be ignored. The cytotoxicity of γ-PGA−DXR to BNL cells was tested in a concentration-dependent manner (100, 300, and 500 ng/mL), shown in Figure 3, panels A (γ-PGA90) and B (γ-PGA1200). The cytotoxicity of DXR was decreased after conjugation to γPGA except for 100 ng/mL of γ-PGA90. The phenomenon was

Figure 2. The cytotoxicity of γ-PGA with a molecular weight of 90 or 1200 kDa to BNL cells after overnight culture. The cell viability was evaluated by MTT assay, and the absorbance was normalized to the control. An error bar represents one standard deviation; n = 4.

more profound at high DXR concentrations. The cell viability for 500 ng DXR/mL dropped to 34.3%, while the cell viability for γPGA−DXR at the same DXR concentration was increased to ∼65% (Figure 3A). Gal conjugation is aimed to enhance the uptake of γ-PGA− DXR into hepatoma cells via binding of the drug conjugates to cell membrane ASGPR. The effect of Gal conjugation on cell death was not obvious for γ-PGA90 at the low DXR conjugation ratio (1 DXR/10 glutamic acids), but became significant at the high DXR conjugation ratio (2 DXR/10 glutamic acids) (Figure 3A). The cell viability for 500 ng/mL γ-PGA90−DXR was 66.5%. Incorporation of Gal further decreased cell viability to 46.8% and 35.0% for γ-PGA90−DXR(2/2) and γ-PGA90− DXR(2/8), respectively, which is comparable with the cytotoxicity of free DXR (Figure 3A). On the other hand, the conjugation of Gal did not seem to affect the cytotoxicity of γPGA1200−DXR (Figure 3B). Since γ-PGA90−DXR possessed better cytotoxic efficacy than γ-PGA1200−DXR, γ-PGA90− DXR(2/0) and γ-PGA90−DXR(2/8) were used for the subsequent experiments. The discrepancy in cytotoxicity of different γ-PGA−DXR−Gal conjugates might be due to the difference in the cellular uptake of DXR, so we next evaluated the cellular uptake of γ-PGA−DXR− Gal conjugates. DXR is an autofluorescent compound, so intracellular distribution of DXR could be observed using fluorescence microscopy. Due to the limited sensitivity of our fluorescent microscope, only DXR uptake from 500 ng DXR/mL or equivalent γ-PGA90−DXR(2/0) and γ-PGA90−DXR−Gal(2/8) could be visualized. The fluorescent images show that red DXR was mainly overlapped with blue nuclei (Figure 4A), indicating that the endocytosed DXR, γ-PGA−DXR and γPGA−DXR−Gal enter cell nuclei. We further quantified the amount of endocytosed DXR from different drug derivatives. 5513

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Figure 3. The viability of BNL cells after incubation with free DXR, γ-PGA−DXR, and γ-PGA−DXR−Gal: (A) 90 kDa and (B) 1200 kDa. An error bar represents one standard deviation; n = 4. @, *, and # denote p < 0.05, 0.01, and 0.001, respectively.

compared with free DXR. Another important aspect of DXR action is that the drug needs to enter cell nuclei since DXR unwinds double-strand DNA by intercalation between layers of base pairs of the double helix, and thus preventing further DNA replication.34 Rodrigues et al. showed that endocytosed PEG− DXR conjugates were primarily localized in the cytoplasm,32 which might explain the lower cytotoxicity of PEG−DXR conjugates compared to free DXR. Differently, we found that γPGA−DXR mainly appeared in the nuclei. The discrepancy between Rodrigues et al.’s and our results may be due to that they used a bioinert polymer, PEG, as the drug carrier, which probably prevent the drug conjugate from entering the nucleus. Furthermore, Rodrigues et al. showed that dissociation of intracellular DXR from PEG is important from the drug’s toxicity.32 On the contrary, DXR conjugated to γ-PGA remained its cell-killing ability in this study. Our results suggest that γPGA−DXR conjugates possess better entrance into nuclei and cytotoxicity compared to DXR conjugated with a bioinert polymer. The conjugation of galactosamine to γ-PGA enhanced the DXR uptake by the hepatoma cells compared to free DXR. The result echoes the conclusion from previous studies.8,35,36 The cytotoxicity of γ-PGA90−DXR was also restored to the level of free DXR after the conjugation of galactosamine. We further evaluated the cytotoxicity of Gal-conjugate γ-PGA−DXR to cell types other than hepatoma cells in order to study the cell selectivity of γ-PGA90−DXR−Gal. Similar to the results on BNL cells, the cytotoxicity of DXR to L929 fibroblasts was decreased after conjugation to γ-PGA (Figure 5). However, unlike the effect to BNL cells, the cytotoxicity of γ-PGA−DXR was not enhanced after Gal conjugation. By comparing the results between BNL cells and L929 cells, we conclude that Gal conjugation increases the cytotoxicity of γ-PGA−DXR on BNL cells but not on L929 cells. Our results indicate that the Gal-functionalized γ-PGA− DXR could enhance the cell specificity of the drug. 3.3. Ultrasound Treatment. The administration of US has been shown to enhance the cytotoxic efficacy of several anticancer drugs such as adriamycin, 4′-O-tetrahydropyranyladriamycin, DXR, and arabinosyl cytosine in vitro.14,37−39 For example, Yoshida et al. showed that a combination of DXR and low-intensity US causes a synergistic enhancement in killing lymphoma cells by ∼100%.14 The synergistic effect by the combination of antidrugs and US has been demonstrated in vivo in animal models.38,40−42 Therefore, in this study γ-PGA−DXR− Gal was combined with US in order to enhance the overall cellkilling efficacy.

Figure 4. Cellular uptake of DXR from 500 ng/mL solution of free DXR, γ-PGA−DXR(2/0), and γ-PGA−DXR−Gal(2/8) after 3 h of incubation. (A) Fluorescent images (blue: nuclei; red: DXR) and (B) the quantification of DXR uptake. An error bar represents one standard deviation; n = 4. *, p < 0.001 vs the other groups.

After 3 h of incubation, the cellular uptake of DXR and γ-PGA− DXR was comparable, but less than the value of γ-PGA−DXR− Gal (Figure 4B, p < 0.05), indicating that Gal conjugation facilitates uptake of γ-PGA−DXR to hepatoma cells. The conjugation of DXR to γ-PGA decreases the drug’s cytotoxicity, consistent with several previous reports regarding other polymer-drug conjugates.29−32 Some studies ascribe the decreased cytotoxicity of polymer-drug conjugates to their increased sizes that limit cellular uptake of drugs to the mechanism of pinocytosis.29,33 However, we did not find a significant decrease in the cellular uptake of γ-PGA90−DXR 5514

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Figure 5. The viability of L929 cells after incubation with free DXR, γPGA90−DXR(2/0), and γ-PGA−DXR90−Gal(2/8). An error bar represents one standard deviation; n = 4. *, p < 0.01 vs γ-PGA−DXR; #, p < 0.001 vs γ-PGA−DXR−Gal.

The US condition applied in this study did not cause the death of BNL cells (Figure 6). The US treatment enhances the cytotoxic effect of free DXR by ∼20% (Figure 6A). Similarly, the cell-killing efficacy of γ-PGA−DXR (Figure 6B) and γ-PGA− DXR−Gal (Figure 6C) was elevated by the US administration. The US-assisted cell-killing effect was more notable at lower DXR doses (e.g., 100 ng/mL). However, the synergistic effect of US was diminished for γ-PGA−DXR−Gal at high DXR concentration (Figure 6C). A patient may benefit from such synergistic enhancement by taking a lower DXR dosage that reduces the side effects of DXR, while receiving equivalent or even improved therapeutic outcomes by US stimulation. Two major theories are proposed to explain the enhanced effect of US on the efficiency of anticancer drugs. First, US enhances cellular uptake of foreign macromolecules via sonoporation43,44 or/and endocytosis.45,46 Therefore, US may increase cellular uptake and intracellular accumulation of anticancer drugs,37,47 thus increasing cell death. However, we did not find a significant increase in the cellular uptake of DXR after US administration (Figure 6D) in spite of the fact that US enhanced the drug’s cytotoxicity. The phenomenon is similar to several previous findings.14,47 Therefore, enhancement of cellular drug uptake via US administration may not explain the increased cell death in this study. Another popular theory ascribes cell damage to the formation of free radicals via sonication, called sonodynamic therapy.48 Ultrasonic cavitation generates free radicals from breakdown of water molecules into hydrogen atoms and hydroxyl radicals, and other radical species, such as hydrogen peroxide, singlet oxygen, and superoxide ions.49 Free radicals can directly deactivate macromolecules, such as proteins and DNA, to impair cellular functions and to damage cell membranes, resulting in the promotion of membrane permeability. Several studies showed that the production of hydroxyl radicals by sonication was enhanced in the presence of DXR, resulting in enhanced cytotoxicity.14,50 We did not determine the generation of free radicals, so we cannot conclude whether the enhancement of cell death is related to sonodynamic effect. Besides the above two mechanisms, another theory suggests that US administration makes cells vulnerable to the attack of anticancer drugs. Nevertheless, the synergistic enhancement by the combination of DXR and US may be a collective effect of several different mechanisms, which requires further investigation.

Figure 6. The viability of BNL cells after incubation with (A) free DXR, (B) γ-PGA90−DXR, and (C) γ-PGA90−DXR−Gal. Solid and blank bars represent the presence and absence of ultrasound treatment, respectively. An error bar represents one standard deviation; n = 3. *, **, and *** represent p < 0.05, 0.01, and 0.001 vs the non-US group, respectively. (D) Cellular uptake of DXR from 500 ng/mL solution of free DXR, γ-PGA90−DXR(2/0), and γ-PGA90−DXR−Gal(2/8) after 3 h of incubation without (blank bars) or with (solid bars) ultrasound treatment. An error bar represents one standard deviation; n = 4. No significant difference was found between US and non-US groups.

4. CONCLUSION In this study, we tried to combine three drug-targeting strategiespolymer−drug conjugate, ligand presentation, and ultrasound administrationto enhance the cytotoxic efficacy and cell selectivity of DXR. We showed that the conjugation of DXR to γ-PGA decreases in vitro cytotoxicity of DXR, while the 5515

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attachment of galactosamine to γ-PGA−DXR restores the cytotoxic efficacy of DXR on hepatoma cells but not on fibroblasts. US treatment enhanced the cell-killing efficacy of γPGA−DXR conjugates. In the future, in vivo evaluation of the stability, biodistribution, and antitumor activity of γ-PGA− DXR−Gal is necessary for potential clinical applications.

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in three human bladder cancer cell lines. Ultrasonics 2007, 46 (1), 68− 73. (14) Yoshida, T.; Kondo, T.; Ogawa, R.; Feril, L.; Zhao, Q.-L.; Watanabe, A.; Tsukada, K. Combination of doxorubicin and lowintensity ultrasound causes a synergistic enhancement in cell killing and an additive enhancement in apoptosis induction in human lymphoma U937 cells. Cancer Chemother. Pharmacol. 2008, 61 (4), 559−567. (15) Marin, A.; Sun, H.; Husseini, G. A.; Pitt, W. G.; Christensen, D. A.; Rapoport, N. Y. Drug delivery in pluronic micelles: effect of highfrequency ultrasound on drug release from micelles and intracellular uptake. J. Controlled Release 2002, 84 (1−2), 39−47. (16) Cochran, M. C.; Eisenbrey, J. R.; Soulen, M. C.; Schultz, S. M.; Ouma, R. O.; White, S. B.; Furth, E. E.; Wheatley, M. A. Disposition of ultrasound sensitive polymeric drug carrier in a rat hepatocellular carcinoma model. Acad. Radiol. 2011, 18 (11), 1341−8. (17) Akao, T.; Kimura, T.; Hirofuji, Y.-s.; Matsunaga, K.; Imayoshi, R.; Nagao, J.-i.; Cho, T.; Matsumoto, H.; Ohtono, S.; Ohno, J.; Taniguchi, K.; Kaminishi, H. A poly(gamma-glutamic acid)-amphiphile complex as a novel nanovehicle for drug delivery system. J. Drug Target 2010, 18 (7), 550−556. (18) Liang, H.-F.; Yang, T.-F.; Huang, C.-T.; Chen, M.-C.; Sung, H.-W. Preparation of nanoparticles composed of poly(γ-glutamic acid)poly(lactide) block copolymers and evaluation of their uptake by HepG2 cells. J. Controlled Release 2005, 105 (3), 213−225. (19) Ramu, A.; Mehta, M. M.; Liu, J.; Turyan, I.; Aleksic, A. The riboflavin-mediated photooxidation of doxorubicin. Cancer Chemother. Pharmacol. 2000, 46 (6), 449−458. (20) Roseman, S.; Daffner, I. Colorimetric method for determination of glucosamine and galactosamine. Anal. Chem. 1956, 28 (11), 1743− 1746. (21) Lin, W. H.; Tsai, W. B. In situ UV-crosslinking gelatin electrospun fibers for tissue engineering applications. Biofabrication 2013, 5, 035008. (22) Roepe, P. D. Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrug resistant cells expressing different levels of P-glycoprotein. Biochemistry 1992, 31 (50), 12555− 12564. (23) Tsai, K. C.; Fang, S. Y.; Yang, S. J.; Shieh, M. J.; Lin, W. L.; Chen, W. S. Time dependency of ultrasound-facilitated gene transfection. J. Gene Med. 2009, 11, 729−736. (24) Lee, J.-L.; Lo, C.-W.; Ka, S.-M.; Chen, A.; Chen, W.-S. Prolonging the expression duration of ultrasound-mediated gene transfection using PEI nanoparticles. J. Controlled Release 2012, 160 (1), 64−71. (25) Akagi, T.; Baba, M.; Akashi, M. Preparation of nanoparticles by the self-organization of polymers consisting of hydrophobic and hydrophilic segments: Potential applications. Polymer 2007, 48 (23), 6729−6747. (26) Hu, F.-Q.; Wu, X.-l.; Du, Y.-Z.; You, J.; Yuan, H. Cellular uptake and cytotoxicity of shell crosslinked stearic acid-grafted chitosan oligosaccharide micelles encapsulating doxorubicin. Eur. J. Pharm. Biopharm 2008, 69 (1), 117−125. (27) Bernard, V.; korpíková, J. i.; Mornstein, V. c.; Slaninová, I. Biological effects of combined ultrasound and cisplatin treatment on ovarian carcinoma cells. Ultrasonics 2010, 50 (3), 357−362. (28) Nasongkla, N.; Shuai, X.; Ai, H.; Weinberg, B. D.; Pink, J.; Boothman, D. A.; Gao, J. cRGD-functionalized polymer micelles for targeted doxorubicin delivery. Angew. Chem. 2004, 116 (46), 6483− 6487. (29) Lee, G. Y.; Park, K.; Nam, J. H.; Kim, S. Y.; Byun, Y. Anti-tumor and anti-metastatic effects of gelatin-doxorubicin and PEGylated gelatin−doxorubicin nanoparticles in SCC7 bearing mice. J. Drug Target 2006, 14 (10), 707−716. (30) Veronese, F. M.; Schiavon, O.; Pasut, G.; Mendichi, R.; Andersson, L.; Tsirk, A.; Ford, J.; Wu, G.; Kneller, S.; Davies, J.; Duncan, R. PEG-doxorubicin conjugates: Influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity. Bioconjugate Chem. 2005, 16 (4), 775−84. (31) Vasey, P. A.; Kaye, S. B.; Morrison, R.; Twelves, C.; Wilson, P.; Duncan, R.; Thomson, A. H.; Murray, L. S.; Hilditch, T. E.; Murray, T.; Burtles, S.; Fraier, D.; Frigerio, E.; Cassidy, J. Phase I clinical and

AUTHOR INFORMATION

Corresponding Authors

*(W.-B.T.) Tel: +886-2-3366-3996; Fax: +886- 2-2362-3040; email: [email protected]. *(W.-S.C.) Tel: +886-2-2312-3456; Fax: +886- 2-2383-2834; email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Science Council, Taiwan, for financial support (Grant Number 100-2923-B-002-004-MY3). REFERENCES

(1) Jang, S. H.; Wientjes, M. G.; Lu, D.; Au, J. L. Drug delivery and transport to solid tumors. Pharm. Res. 2003, 20 (9), 1337−1350. (2) Matsumura, Y.; Maeda, H. A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic accumulation of proteins and the antitumor agent SMANCS. Cancer Res. 1986, 1986, 6387−6392. (3) Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv. Drug Delivery Rev. 2011, 63 (3), 136−151. (4) Li, C.; Wallace, S. Polymer−drug conjugates: Recent development in clinical oncology. Adv. Drug Delivery Rev. 2008, 60 (8), 886−98. (5) Dubowchik, G. M.; Walker, M. A. Receptor-mediated and enzymedependent targeting of cytotoxic anticancer drugs. Pharmacol. Ther. 1999, 83 (2), 67−123. (6) Garnett, M. C. Targeted drug conjugates: Principles and progress. Adv. Drug Delivery Rev. 2001, 53 (2), 171−216. (7) Tol, J.; Koopman, M.; Cats, A.; Rodenburg, C. J.; Creemers, G. J. M.; Schrama, J. G.; Erdkamp, F. L. G.; Vos, A. H.; van Groeningen, C. J.; Sinnige, H. A. M.; Richel, D. J.; Voest, E. E.; Dijkstra, J. R.; Vink-Borger, M. E.; Antonini, N. F.; Mol, L.; van Krieken, J. H. J. M.; Dalesio, O.; Punt, C. J. A. Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer. N. Engl. J. Med. 2009, 360, 563−572. (8) Liang, H.-F.; Chen, C.-T.; Chen, S.-C.; Kulkarni, A. R.; Chiu, Y.-L.; Chen, M.-C.; Sung, H.-W. Paclitaxel-loaded poly(γ-glutamic acid)− poly(lactide) nanoparticles as a targeted drug delivery system for the treatment of liver cancer. Biomaterials 2006, 27 (9), 2051−2059. (9) Lai, C.-H.; Lin, C.-Y.; Wu, H.-T.; Chan, H.-S.; Chuang, Y.-J.; Chen, C.-T.; Lin, C.-C. Galactose encapsulated multifunctional nanoparticle for HepG2 cell internalization. Adv. Funct. Mater. 2010, 20 (22), 3948− 3958. (10) Seymour, L. W.; Ferry, D. R.; Anderson, D.; Hesslewood, S.; Julyan, P. J.; Poyner, R.; Doran, J.; Young, A. M.; Burtles, S.; Kerr, D. J. Cancer research campaign phase, I. I. I. C. T. c. hepatic drug targeting: Phase I evaluation of polymer-bound doxorubicin. J. Clin. Oncol. 2002, 20 (6), 1668−76. (11) Husseini, G. A.; Pitt, W. G. Micelles and nanoparticles for ultrasonic drug and gene delivery. Adv. Drug Delivery Rev. 2008, 60 (10), 1137−1152. (12) Tamura, T.; Nishi, T.; Goto, T.; Takeshima, H.; Dev, S. B.; Ushio, Y.; Sakata, T. Intratumoral delivery of interleukin 12 expression plasmids with in vivo electroporation is effective for colon and renal cancer. Hum. Gene Ther. 2001, 12 (10), 1265−76. (13) Arthur, C.; Flaig, T.; Su, L.-J.; Denney, R.; Barnes, F.; Glodé, L. M. The effect of ultrasonic irradiation on doxorubicin-induced cytotoxicity 5516

dx.doi.org/10.1021/la500352g | Langmuir 2014, 30, 5510−5517

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Article

pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: First member of a new class of chemotherapeutic agents-drug-polymer conjugates. Cancer Research Campaign Phase I/II Committee. Clin. Cancer Res. 1999, 5 (1), 83−94. (32) Rodrigues, P. C. A.; Beyer, U.; Schumacher, P.; Roth, T.; Fiebig, H. H.; Unger, C.; Messori, L.; Orioli, P.; Paper, D. H.; Mülhaupt, R.; Kratz, F. Acid-sensitive polyethylene glycol conjugates of doxorubicin: Preparation, in vitro efficacy and intracellular distribution. Bioorg. Med. Chem. 1999, 7 (11), 2517−2524. (33) Duncan, R.; Kopeckova-Rejmanova, P.; Strohalm, J.; Hume, I.; Cable, H. C.; Pohl, J.; Lloyd, J. B.; Kopecek, J. Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of daunomycin and puromycin conjugates in vitro. Br. J. Cancer 1987, 55 (2), 165−74. (34) Al-Ghamdi, S. S. Time and dose dependent study of doxorubicin induced DU-145 cytotoxicity. Drug Metab. Lett. 2008, 2 (1), 47−50. (35) Liang, H.-F.; Chen, S.-C.; Chen, M.-C.; Lee, P.-W.; Chen, C.-T.; Sung, H.-W. Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system against cultured HepG2 cells. Bioconjugate Chem. 2006, 17 (2), 291−299. (36) Hashida, M.; Akamatsu, K.; Nishikawa, M.; Yamashita, F.; Takakura, Y. Design of polymeric prodrugs of prostaglandin E1 having galactose residue for hepatocyte targeting. J. Controlled Release 1999, 62 (1−2), 253−262. (37) Yu, T.; Wang, Z.; Jiang, S. Potentiation of cytotoxicity of adriamycin on human ovarian carcinoma cell line 3AO by low-level ultrasound. Ultrasonics 2001, 39 (4), 307−9. (38) Tachibana, K.; Uchida, T.; Tamura, K.; Eguchi, H.; Yamashita, N.; Ogawa, K. Enhanced cytotoxic effect of Ara-C by low intensity ultrasound to HL-60 cells. Cancer Lett. 2000, 149 (1−2), 189−94. (39) Yumita, N.; Kaneuchi, M.; Okano, Y.; Nishigaki, R.; Umemura, K.; Umemura, S. Sonodynamically induced cell damage with 4′-Otetrahydropyranyladriamycin, THP. Anticancer Res. 1999, 19 (1A), 281−4. (40) Sur, P.; Ghosh, P.; Bag, S. P.; Sur, B.; Chatterjee, S. N. On the inhibitory activities of a new boron compound and ultrasound against the mouse ascites tumour. Chemotherapy 1999, 45 (5), 360−9. (41) Tomizawa, M.; Ebara, M.; Saisho, H.; Sakiyama, S.; Tagawa, M. Irradiation with ultrasound of low output intensity increased chemosensitivity of subcutaneous solid tumors to an anti-cancer agent. Cancer Lett. 2001, 173 (1), 31−5. (42) Nelson, J. L.; Roeder, B. L.; Carmen, J. C.; Roloff, F.; Pitt, W. G. Ultrasonically activated chemotherapeutic drug delivery in a rat model. Cancer Res. 2002, 62 (24), 7280−3. (43) Bekeredjian, R.; Chen, S.; Grayburn, P. A.; Shohet, R. V. Augmentation of cardiac protein delivery using ultrasound targeted microbubble destruction. Ultrasound Med. Biol. 2005, 31 (5), 687−91. (44) Chen, S.; Ding, J. H.; Bekeredjian, R.; Yang, B. Z.; Shohet, R. V.; Johnston, S. A.; Hohmeier, H. E.; Newgard, C. B.; Grayburn, P. A. Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (22), 8469−74. (45) Meijering, B. D.; Juffermans, L. J.; van Wamel, A.; Henning, R. H.; Zuhorn, I. S.; Emmer, M.; Versteilen, A. M.; Paulus, W. J.; van Gilst, W. H.; Kooiman, K.; de Jong, N.; Musters, R. J.; Deelman, L. E.; Kamp, O. Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation. Circ. Res. 2009, 104 (5), 679−87. (46) Paula, D. M.; Valero-Lapchik, V. B.; Paredes-Gamero, E. J.; Han, S. W. Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis. J. Gene Med. 2011, 13 (7−8), 392−401. (47) Loverock, P.; ter Haar, G.; Ormerod, M. G.; Imrie, P. R. The effect of ultrasound on the cytotoxicity of adriamycin. Br. J. Radiol. 1990, 63 (751), 542−6. (48) Yu, T.; Wang, Z.; Mason, T. J. A review of research into the uses of low level ultrasound in cancer therapy. Ultrason. Sonochem. 2004, 11 (2), 95−103. (49) Kardos, N.; Luche, J. L. Sonochemistry of carbohydrate compounds. Carbohydr. Res. 2001, 332 (2), 115−31.

(50) Umemura, S.; Yumita, N.; Okano, Y.; Kaneuchi, M.; Magario, N.; Ishizaki, M.; Shimizu, K.; Sano, Y.; Umemura, K.; Nishigaki, R. Sonodynamically-induced in vitro cell damage enhanced by adriamycin. Cancer Lett. 1997, 121 (2), 195−201.

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