Biomaterials 35 (2014) 1735e1743

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Hypoxia-responsive polymeric nanoparticles for tumor-targeted drug delivery Thavasyappan Thambi a, b, V.G. Deepagan a, b, Hong Yeol Yoon a, b, c, Hwa Seung Han a, b, Seol-Hee Kim d, Soyoung Son a, g, Dong-Gyu Jo d, g, Cheol-Hee Ahn e, Yung Doug Suh b, f, Kwangmeyung Kim c, Ick Chan Kwon c, Doo Sung Lee a, b, Jae Hyung Park a, b, f, g, * a

Department of Polymer Science and Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea d School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea e Department of Materials Science and Engineering, Seoul National University, Seoul 151-744, Republic of Korea f NanoBio Fusion Research Center, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea g Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Suwon 440-746, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 October 2013 Accepted 7 November 2013 Available online 26 November 2013

Hypoxia is a condition found in various intractable diseases. Here, we report self-assembled nanoparticles which can selectively release the hydrophobic agents under hypoxic conditions. For the preparation of hypoxia-responsive nanoparticles (HR-NPs), a hydrophobically modified 2-nitroimidazole derivative was conjugated to the backbone of the carboxymethyl dextran (CM-Dex). Doxorubicin (DOX), a model drug, was effectively encapsulated into the HR-NPs. The HR-NPs released DOX in a sustained manner under the normoxic condition (physiological condition), whereas the drug release rate remarkably increased under the hypoxic condition. From in vitro cytotoxicity tests, it was found the DOXloaded HR-NPs showed higher toxicity to hypoxic cells than to normoxic cells. Microscopic observation showed that the HR-NPs could effectively deliver DOX into SCC7 cells under hypoxic conditions. In vivo biodistribution study demonstrated that HR-NPs were selectively accumulated at the hypoxic tumor tissues. As consequence, drug-loaded HR-NPs exhibited high anti-tumor activity in vivo. Overall, the HRNPs might have a potential as nanocarriers for drug delivery to treat hypoxia-associated diseases. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Hypoxia Nanoparticles 2-Nitroimidazole Bioreduction Drug delivery

1. Introduction Hypoxia, a pathological condition in which tissue is deprived of a supply of adequate oxygen, is a hallmark of various intractable diseases such as cancer, cardiopathy, ischemia, rheumatoid arthritis, and vascular diseases [1,2]. For example, experimental and clinical studies have demonstrated that tissue partial pressures of oxygen, measured from ischemic stroke and cancer patients, are near zero mm Hg, which is substantially lower than in normal tissue (w30 mm Hg) [2,3]. Since hypoxia is involved in many aspects of the biology of such diseases, it significantly affects therapeutic responses. In particular, hypoxia is a negative factor for cancer therapy

* Corresponding author. School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea. Tel.: þ82 31 290 7288; fax: þ82 31 290 7309. E-mail addresses: [email protected], [email protected] (J.H. Park). 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.11.022

as it contributes to chemoresistance, radioresistance, angiogenesis, invasiveness, and metastasis [4]. Nevertheless, owing to its unique features, which are rarely seen in normal tissue, hypoxia is emerging as a primary target in the development of diagnostic agents and therapeutic drugs. Representative approaches for hypoxia-targeted cancer therapies are based on regulation of hypoxia inducible factor-1 [5] and on the use of bioreductive prodrugs that can be activated in the reductive environment of hypoxia [2,4]. For hypoxia imaging, many nitroaromatic or quinone derivatives with hypoxiaresponsive moieties have been employed in the molecular design of diagnostic agents [6e9]. Of the derivatives investigated to date, 2-nitroimidazoles (NIs) have been most widely utilized in the development of imaging agents and bioreductive prodrugs because of their high sensitivity to hypoxia [10,11]. It has been demonstrated that, under hypoxic conditions, NIs are converted to hydrophilic 2-aminoimidazoles via a series of selective bioreductions, which are highly reactive to macromolecules in hypoxic tissues [11,12]. There,

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however, are no literature available for targeted drug delivery systems using hypoxia-responsive nanoparticles. Self-assembled polymeric nanoparticles composed of amphiphilic polymers have emerged as promising nanocarriers for various anticancer drugs [13e16]. They exhibit unique characteristics as drug carriers, including enhancement of drug solubility, high thermodynamic stability, and preferential accumulation in tumor tissue via the enhanced permeation and retention (EPR) effect [14,17]. Conventional nanocarriers, however, often show limited anti-tumor efficacy because they release the drug in a sustained manner even at the target site of action [18]. Polymeric materials that respond to the pathophysiological conditions in tumors have been recently utilized to construct nanoparticles for drug delivery with enhance therapeutic efficacy [19,20]. Such stimuli-responsive nanocarriers are expected to reach the tumor site via the EPR effect and release the drug rapidly when they are exposed to tumor tissue [21]. To date, many stimuli for the development of smart nanocarriers have been explored, including ultraviolet radiation [22,23], glutathione [24e26], pH [27e29], and temperature [30e32]. Some of these stimuli-responsive drug carriers have advanced to clinical trials [33]. 2. Materials and methods 2.1. Materials Carboxymethyl-dextran sodium salt (CM-Dex, Mn ¼ 10000e20000 Da), 1-ethyl3(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2nitroimidazole (NI), doxorubicin$hydrochloride (DOX$HCl), 1.25 M HCl in methanol, and 6-(Boc-amino)hexyl bromide were purchased from SigmaeAldrich Co. (St. Louis, MO, USA). The water used in the experiments was prepared by an AquaMaxUltra water purification system (Younglin Co., Anyang, Korea). All other chemicals were of analytical grade and used without further purification.

2.2. Synthesis of hypoxia-responsive conjugate In this study, we attempted to prepare hypoxia-responsive nanoparticles (HRNPs) that allow for the facilitated release of hydrophobic drugs at hypoxic tumor tissue (Fig. 1). In order to prepare the amphiphilic conjugate which can form HR-NPs in an aqueous condition, the NI derivative was chemically conjugated to the backbone of water-soluble CM-Dex through amide formation (Fig. 2). First, NI was converted to 6-(2-nitroimidazole)hexylamine for reaction with the carboxylic acids of CM-Dex (Fig. S1). In brief, NI (0.6 g, 5.3 mmol) was dissolved in DMF, to which K2CO3 (1.1 g, 7.95 mmol) was added. 6-(Boc-amino)hexyl bromide (1.56 g, 5.57 mmol) in DMF was then added dropwise and stirred at room temperature (RT) overnight. The reaction mixture was filtered and washed with methanol, after which the residual solvent was evaporated. The solid obtained was suspended in water and extracted with ethyl acetate. The organic layer was separated, dried over sodium sulfate, and concentrated to obtain the product for step 1. The resulting product was dissolved in methanol and cooled to 0  C, to which 10 ml of 1.25 M HCl in methanol was added and stirred at RT for 24 h. The solvent was removed from the reaction mixture using a rotary evaporator. The crude solid was recrystallized from ethanol to obtain amine-functionalized 2-nitroimidazole (NI derivative). Next, the NI derivative was conjugated to CM-Dex in the presence of EDC and NHS (Fig. 2). In brief, CM-Dex (0.2 g, 0.9 mmol) was dissolved in a 1:1 mixture of formamide and dimethyl formamide, after which EDC (0.6e2.07 g, 3.6e10.88 mmol) and NHS (0.41e1.24 g, 3.6e10.88 mmol) were added and stirred for 15 min. The NI derivative (0.19e0.573 g, 0.9e2.7 mmol) in DMF was slowly added to the reaction mixture and stirred for 1 day. The resulting solution was dialyzed against an excess of water/methanol (1/3e1/1 v/v) for 1 day and against distilled water for 2 days before being lyophilized. The amount of NI derivative to CM-Dex was spectrophotometrically determined from the characteristic peak of the NI derivative at 325 nm using a UVevis spectrophotometer (Optizen 3220UV, Mecasys Co., Ltd., Daejeon, Korea). For cellular experiments and animal tests, Cy5.5-labeled hypoxia-responsive nanoparticles (Cy5.5-HR-NPs) were prepared, as previously described [17]. 2.3. Characterization The chemical structures of the NI derivatives and the conjugate were characterized using 1H NMR (JNM-AL300, JEOL, Tokyo, Japan) operating at 300 MHz, for which the samples were dissolved in CD3OD, D2O, or DMSO-D6. The sizes of the

Fig. 1. Schematic illustration of the formation of drug-loaded HR-NPs and in vivo tumor-targeting pathways. The HR-NPs can reach the tumor site via the EPR effect, followed by intracellular drug release at hypoxic tissue.

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Loading efficiency ð%Þ ¼ ðweight of loaded drug=weight of drug in feedÞ  100%

O OH OH O

O

O

O OH OH

OH

O

Loading content ð%Þ ¼ ðweight of loaded drug=weight of polymerÞ  100%

O

O OH OH

O

O

OH

O

OH n

EDC/NHS NI derivative

O OH OH O

O

OH

O

O OH OH

O

O

2.5. Cytotoxicity and intracellular drug release

O

O OH OH

O

NH

O

The loading efficiency and content of DOX into the DOX-HR-NPs was found to be 76% and 7.6 wt%, respectively. For the release experiment, DOX-HR-NPs were dispersed in PBS (pH 7.4) and the solution was transferred to cellulose membrane tubes (MWCO ¼ 3500 Da). Thereafter, the dialysis tube was immersed in degassed PBS (pH 7.4) with 100 mM NADPH and degassing with nitrogen was continued for the entire period of the release experiment to maintain the hypoxic condition [36,37]. For the control experiment (normoxic), the sample was immersed in PBS (pH 7.4) containing 100 mM NADPH without degassing. Each sample was gently shaken at 37  C and 100 rpm. The medium was refreshed at predetermined time intervals, and the DOX concentration was measured using a UVevis spectrophotometer at 485 nm.

OH n

The SCC7 (squamous carcinoma) cell line, obtained from the American Type Culture Collection (Rockville, MD, USA), was cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 10% (v/v) fetal bovine serum and 1% (w/v) penicillin-streptomycin at 37  C in a humidified 5% CO2-95% air atmosphere. The cells were seeded at a density of 1  104 cells/well in 96-well flat-bottomed plates. After 1 day of growth, the cells were washed twice with PBS (pH 7.4) and incubated for 24 h with various concentrations of free DOX or DOX-HR-NPs under the hypoxic or normoxic condition. The cells were then washed twice with PBS (pH 7.4) and fresh culture medium was added. Twenty microliters of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide solution (5 mg/ml in PBS) was added to each well, and the cells were incubated for an additional 4 h at 37  C. Subsequently, the medium was removed and the cells were dissolved in DMSO. The absorbance at 570 nm was measured using a microplate reader (BioTek, Seoul, Korea). The statistical significance of differences (p < 0.05) between groups tested was determined using an unpaired student’s t test. To investigate intracellular drug release from the HR-NPs, cells were incubated with DOX-HR-NPs under hypoxic or normoxic conditions for 12 h. The cells were then washed twice with PBS (pH 7.4) and fixed with 4% formaldehyde solution. For nuclear staining, the cells were incubated with 4,6-diamino-2-phenylinodole (DAPI) for 10 min at RT, followed by washing with PBS (pH 7.4). The intracellular localization of DOX released from HR-NPs was monitored using an IX81-ZDC focus drift compensating microscope (Olympus, Tokyo, Japan).

2.6. In vivo biodistribution of HR-NPs and tissue staining

N

NO2 N

Fig. 2. Synthetic route for preparation of the hypoxia-responsive CM-Dex conjugates.

particles were determined at 25  C using a FPAR-1000 fiber optics particle analyzer (Otsuka Electronics, Osaka, Japan). The morphology of the particles was observed using by transmission electron microscopy (TEM, Philips CM30) at an accelerating voltage of 200 ekV. For TEM images, samples were dispersed in distilled water and dropped on a 200 mesh copper grid. All samples were treated with 1% uranyl acetate for negative staining. The zeta potential (z) was measured using a zetasizer (90 PLUS, BrookHAVEN Instruments Cooperation, New York, USA). The hypoxic condition was maintained using a CO2/O2 incubator (Vision Scientific Co., Ltd, Korea). 2.4. Drug loading and release of DOX from HR-NPs DOX was loaded into the nanoparticles by the emulsion method [29]. In brief, DOX$HCl (3 mg) was dissolved in chloroform containing 3.0 equimolar amount of triethylamine. The resulting solution was added to the aqueous solution containing HR-NPs (30 mg), leading to formation of an oil-in-water emulsion. This emulsion was kept in dark conditions overnight with stirring to allow evaporation of the chloroform. The solution was then dialyzed against an excess amount of distilled water for 24 h to remove unloaded DOX, followed by lyophilization to obtain DOXHR-NPs. The loading efficiency and content of DOX in the DOX-HR-NPs were determined using a UVevis spectrophotometer (Optizen 3220UV, Mecasys Co., Ltd., Daejeon, Korea) at 485 nm. For this experiment, DOX-HR-NPs were dissolved in a DMSO/water (1v/1v) mixture, and the calibration curve was obtained using DMSO/ water (1v/1v) solutions with different DOX concentrations. The loading efficiency and loading content of DOX were calculated using the following formulas:

The tumor-bearing mice were prepared by injecting a suspension of 1  106 SCC7 cells in physiological saline (100 ml) into the subcutaneous dorsa of athymic nude mice (7 weeks old, 20e25 g). After 14 days of subcutaneous inoculation, Cy5.5HR-NPs were injected into the tail vein of the tumor-bearing mice at a dose of 5 mg/ kg. The biodistribution of HR-NPs were evaluated as a function time using eXplore Optix system (ART Advanced Research Technologies Inc., Montreal, Canada). Laser power and count time settings were optimized at 15 mW and 0.3 s per point. Excitation and emission spots were raster-scanned in 1 mm steps over the selected region of interest. A 670 nm-pulsed laser diode was used to excite the Cy5.5 molecules. The fluorescence emission at 700 nm was collected and detected through the fast photomultiplier tube (Hammamatsu, Japan) and a time-correlated single photon counting system (Becker and Hickl Gmbh, Berlin, Germany), respectively. The major organs and tumors were dissected from SCC7 tumor-bearing mice at 24 h after intravenous injection of Cy5.5-HR-NPs. NIR fluorescence images of dissected organs and tumors were obtained with a 12-bit CCD camera (Kodak Image Station 4000 MM, New Haven, CT) equipped with a special C-mount lens and Cy5.5 bandpass emission filter (680 nme720 nm; Omega Optical). The tissue distribution of HRNPs was quantified by measuring the NIR fluorescence intensity at the region of interest. All values are expressed as means  SD for groups of five animals. For hypoxic tissue staining, pimonidazole $ hydrochloride (HypoxyprobeÔ-1) as a hypoxic staining probe was used. Pimonidazole is activated in hypoxic cells and subsequently form covalent adduct with thiol-containing proteins, peptides and amino acids. The adduct can be stained using fluorescently labeled monoclonal antibody (Mab1). Pimonidazole $ hydrochloride (100 mg/kg) was intravenously administrated into the tail vein of SCC7 tumor-bearing mice, 30 min later Cy5.5-HRNPs (5 mg/kg) was injected and allowed for 1 h. Finally, Hoechst 33342 was injected and allowed for 10 min to label cell nuclei. For hypoxic tissue staining, tumor tissues were removed from the sacrificed mice, fixed with 2% paraformaldehyde solution, and embedded in paraffin. Frozen tissues were sectionalized from 10 to 20 mm using a Cryostat Microtome (CM1850, Leica Microsystems Nussloch GmbH, Germany). Frozen sections were transferred to 4% paraformaldehyde for fixation at 4  C for 20 min. Fixed slides were washed in PBS and incubated with methanol at 20  C for 10 min, and blocked with PBS containing 1% bovine serum albumin. Pimanidazole adducts were detected with FITC-conjugated IgG1 mouse monoclonal antibodies (clone 4.3.11.3, Hypoxyprobe, Inc. Burlington, MA, USA) at 1:400 dilution for 1 h. Finally, the stained sections were added with mounting solution, covered with a

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cover slip, and analyzed with a confocal microscope (LSM 700, Carl Zeiss Micro Imaging GmbH, Germany) equipped with a 40 water-emersion objective.

2.7. In vivo anti-tumor efficacy of DOX-HR-NPs To evaluate the anti-tumor efficacy of HR-NPs, SCC7 tumor-bearing mice were prepared, as previously described. Mice were divided into three groups: (i) normal saline (ii) free DOX at 5 mg/kg, and (iii) DOX-HR-NPs at 5 mg DOX/kg. When tumors reached 8 mm in diameter, each sample was injected once every three days. Tumor volumes were calculated as a  b2/2, were a was the largest and b the smallest diameter. The statistical significance of differences (p < 0.05) between groups tested was determined using one-way ANOVA test.

3. Results and discussion 3.1. Synthesis and characterization of HR-NPs In order to prepare hypoxia-responsive amphiphilic conjugates that are capable of self-assembly in physiological solution, a NI derivative was conjugated to the backbone of carboxymethyl dextran (CM-Dex) through the formation of an amide bond. CMDex was chosen as the hydrophilic backbone of the conjugate as it has been widely used as drug carriers, primarily due to its biocompatibility and biodegradability [34,35]. The NI derivative was synthesized via the two-step process, in which the hexyl amino group was introduced to the secondary amine of NI.

The detailed synthetic procedure used to prepare the NI derivative, called (6-(2-nitroimidazole)hexylamine), and 1H NMR spectra demonstrating the chemical structure are shown in Supporting Information (Figs. S1 and S2). The chemical structure of the conjugate was also confirmed using 1H NMR (Fig. 3). During the synthetic procedure, the degree of substitution (DS), defined as the number of the NI derivatives per 100 CM-Dex sugar residues, was controlled by varying the molar feed ratio of the primary amino group in the NI derivative to the carboxyl group in CM-Dex. As expected, the DS increased as the feed ratio increased (Table 1). The prepared conjugates were coded depending on the DS of the NI derivative. For example, HR-NP8 indicates conjugate with a DS value of 8. From size measurements, we found that self-assembled nanoparticles were constructed with conjugates with a DS value higher than 7. Conjugates with lower DS values did not form nanoparticles, possibly because of their high hydrophilicity. The mean diameters of the HRNPs were in the range of 176e192 nm, depending on the DS value. The zeta potentials of all the HR-NPs were negative, indicating that nanoparticle surfaces were covered by hydrophilic CM-Dex. Since the NI derivative was conjugated to the carboxyl group of CM-Dex, HR-NP11 exhibited a lower zeta potential value than HR-NP8. Transmission electron microscopy (TEM) revealed that the nanoparticles were spherical in shape (Fig. 4). The HR-NPs were maintained at their initial size for at least 5 days in physiological solution (pH 7.4) (Fig. S3), suggesting that the nanoparticles had high stability.

Fig. 3. 1H NMR spectra of the conjugate. The sample was dissolved in D2O:CD3OD (1v/:1v).

T. Thambi et al. / Biomaterials 35 (2014) 1735e1743 Table 1 Physicochemical characteristics of HR-NPs. Samplea

FRb

DSc

Size (nm)d

Xe

HR-NP1 HR-NP3 HR-NP8 HR-NP11

0.2 0.4 1.0 2.0

1.86 3.35 7.99 11.76

e e 176.38  3.55 192.23  3.42

1.65 2.98 7.11 10.47

a

HR-NPs with different DS values. Molar feed ratio of the NI derivative to the CM-Dex sugar residue. c Degree of substitution of the NI derivative in a CM-Dex molecule was determined by the UVevis absorbance at 325 nm. d Mean diameters were measured using a particle analyzer. e Weight percentage of the NI derivative in the conjugate. b

The sensitivity of the HR-NPs to hypoxia was assessed by measuring the change in the absorption peaks after incubation in PBS (pH 7.4) under normoxic (20% O2, 5% CO2) or hypoxic (0.1% O2, 5% CO2) conditions for 3 h at 37  C (Fig. 5a). No significant changes in the absorption peaks of the HR-NPs were observed under normoxic conditions. Interestingly, under hypoxic conditions, the characteristic peak of NI at 325 nm completely disappeared, whereas a new peak appeared at 278 nm, corresponding to the characteristic peak of 2-aminoimidazole. This is consistent with previous results showing that the nitro group of NI is converted to an amino group under low oxygen conditions [3]. This classical

20

20

a

b

HR-NP8

HR-NP11

15 Intensity (%)

15

Intensity (%)

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10

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5

0

0 0

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700

0

800

100

200

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400

500

600

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800

Size (nm)

Size (nm)

Fig. 4. (a) and (b) size distribution of HR-NPs. Insets are for TEM images of HR-NPs.

-25

a

1.4

HR-NP8 (Normoxic) HR-NP11 (Normoxic) HR-NP8 (Hypoxic) HR-NP11 (Hypoxic)

Absorbance

1.2 1.0

NH2 NADPH

N

0.8

N

0.6

6e-1

N

b Normoxic Hypoxic

-20

NO2 N

Zeta potential (mV)

1.6

-15

-10

0.4

-5 0.2 0.0 260

280

300 320 340 360 Wavelength (nm)

380

400

0 HR-NP8

HR-NP11

Fig. 5. (a) Absorption spectra of HR-NPs incubated under hypoxic and normoxic conditions for 3 h. Measurements were performed in PBS buffer containing 100 mM NADPH as an electron donor (The illustration shows the conversion of NI into 2-aminoimidazole). (b) The zeta potential of HR-NPs incubated under hypoxic and normoxic conditions with 100 mM NADPH as an electron donor (mean  SD, n ¼ 3).

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higher for HR-NP11 because of the larger amount of NI derivative in the conjugate. It should be emphasized that these structural changes under hypoxia may affect the hydrophilicity of HR-NPs as the primary amino group in the conjugate is relatively hydrophilic, compared to the nitro group. Indeed, NI is poorly soluble in water, whereas its reductive derivative, 2-aminoimidazole, is highly water-soluble. Based on this unique feature of the NI derivative, we hypothesized that HR-NPs can selectively release the hydrophobic drugs at the hypoxic condition.

140 DOX-HR-NPs (Normoxic) DOX-HR-NPs (Hypoxic)

Cumulative DOX release (%)

120 100 80

3.2. Drug release behavior

60 40 20 0 0

2

4

6

8

10

12

Time (h) Fig. 6. In vitro DOX release behavior of HR-NPs under hypoxic and normoxic conditions. Measurements were performed in PBS buffer (pH 7.4) containing 100 mM NADPH as an electron donor (mean  SD, n ¼ 3).

To evaluate the effect of hypoxia on drug release behavior, doxorubicin (DOX) as an anticancer drug was encapsulated into HRNP11 via the emulsion method. DOX-loaded HR-NPs (DOX-HR-NPs) were incubated in hypoxic or normoxic conditions and the DOX release from HR-NPs was quantitatively analyzed as a function of time (Fig. 6). As expected, the release rate of DOX from HR-NPs was significantly higher in hypoxic conditions than in normoxic conditions. The results showed that DOX was released from HR-NPs in a sustained manner under normoxic conditions, resulting in only a 49% release of DOX over 12 h. Notably, DOX was completely released within 12 h under hypoxic conditions. This demonstrates that HR-NPs can selectively release DOX under hypoxic conditions, primarily owing to bioreduction of the hydrophobic NI derivative to the hydrophilic derivative. 3.3. In vitro cytotoxicity and intracellular drug release

reduction of nitro to amine occurs via the transfer process of six electrons, involving nitroso (eN]O) and hydroxylamino (eNHOH) intermediates [12]. The structural change in the conjugates was further confirmed by measuring their zeta potentials (Fig. 5b). No significant changes in the zeta potentials were observed when HRNPs were incubated under normoxic conditions. However, the zeta potentials of HR-NPs significantly decreased under hypoxic condition due to the presence of positively charged amino groups. In particular, the extent of the decrease in the zeta potential was much

To evaluate the in vitro cytotoxicity of HR-NPs, SCC7 cells were treated with HR-NPs for 24 h at 37  C and cell viability was measured using the MTT assay (Fig. 7a). HR-NPs showed no significant cytotoxicity to SCC7 cells up to a polymer concentration of 100 mg/ml. The cytotoxicities of DOX-HR-NPs and free DOX were also evaluated in normoxic and hypoxic conditions (Fig. 7b). The cytotoxicity of free DOX was similar in both conditions. On the other hand, DOX-HR-NPs showed significantly higher cytotoxicity

120

140

a

b

HR-NP8 HR-NP11 100

120 100

Cell viability (%)

Cell viability (%)

DOX-HR-NPs (Normoxic) DOX-HR-NPs (Hypoxic) Free DOX (Normoxic) Free DOX (Hypoxic)

80 60

80

* *

60

*

40 40

20 0

0 10

25

50

100

Polymer concentration ( g/ml)

20 30 40 DOX concentration ( g/ml)

Fig. 7. (a) In vitro cytotoxicity of bare HR-NPs incubated with SCC7 cells for 24 h (mean  SD, n ¼ 3). (b) Dose-dependent cytotoxicity of DOX-HR-NPs and free DOX. Samples were incubated with SCC7 cells under hypoxic and normoxic conditions for 24 h (mean  SD, n ¼ 3). Asterisks (*) denote statistically significant differences (p < 0.05 compared with DOXloaded sample incubated under normoxic condition).

T. Thambi et al. / Biomaterials 35 (2014) 1735e1743

a

DAPI

DOX

DIC

1741

Overlay

b

Fig. 8. Intracellular release of DOX from HR-NPs. Samples were incubated with SCC7 cells under (a) normoxic and (b) hypoxic conditions.

Fig. 9. In vivo non-invasive fluorescence imaging of HR-NPs in tumor-bearing mice. (a) Time-dependant whole body image of athymic nude mice bearing SCC7 tumors after intravenous injection of Cy5.5-HR-NPs. (b) Ex vivo fluorescence image of normal organs and tumor tissues collected at one day post-injection of HR-NPs. (c) Quantification of HR-NPs in normal organs and tumor tissue. Error bars represent the standard deviation for five animals per group. (d) Histological staining of hypoxic tumor tissue. FITC-labeled monoclonal antibody was used for staining hypoxic tissue.

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7

14000

a 12000

6 5 Tumor weight (g)

10000 8000 6000

Bar: 10mm

Tumor volume (mm3)

b

Saline Free DOX (5mg/kg) DOX-HR-NPs (5mg DOX/kg)

4000 2000

4 3 2 1

*

0

0 0

5

10

15 20 Time (day)

25

Saline

30

DOX

DOX-HR-NPs

Fig. 10. Antitumor efficacy of DOX-HR-NPs. (a) Tumor growth of SCC7 cancer xenografts treated with saline, free DOX, and DOX-HR-NPs at a DOX dose of 5 mg/kg. Insets are the representative tumor images excised at 16 days post-treatment. (b) Tumor weights at 16 days post-treatment. Error bars represent standard deviation for five animals per group. Asterisks (*) denote statistically significant differences (p < 0.05) calculated by an one-way Anova test.

in hypoxic conditions than in normoxic conditions. This might be due to the rapid release of DOX from HR-NPs under the reduced oxygen concentration, as shown in Fig. 6. To verify intracellular drug release, the DOX-HR-NPs were monitored using CLSM after incubation with SCC7 cells (Fig. 8). After 12 h of incubation with DOX-HR-NPs under hypoxic conditions, strong fluorescence was observed in the cytoplasm of the cells, indicating rapid release of DOX from the nanoparticles. On the other hand, cells incubated under normoxic conditions showed only a weak fluorescent signal in the cytoplasm of the cells. These results are in agreement with the in vitro release behavior (Fig. 6). Overall, it is evident that the hypoxia-responsive DOX-HR-NPs prepared in this study have the capability for effective delivery of hydrophobic drugs into hypoxic cells. 3.4. In vivo biodistribution and tumor targeting characteristics of HR-NPs Although numerous nanoparticles showed excellent properties as the drug carrier in vitro, clinical applications have been often limited by undesirable biodistribution and poor tumor targetability [38]. To evaluate tumor targetability of HR-NPs, their in vivo biodistribution were assessed using a real-time near-infrared fluorescence (NIRF) imaging technique, following systemic administration of Cy5.5-labeled HR-NPs into the tail vein of SCC7 tumor-bearing mice. The significant NIRF signals were found in the whole body for up to 12 h, suggesting prolonged circulation of HRNPs in blood (Fig. 9a). Interestingly, the strong NIRF signal at the tumor site was observed only at 1 h post-injection, which was maintained for up to 12 h. The ex vivo image of the tissues, retrieved at 24 h postinjection, supported high tumor targetability of HR-NPs (Fig. 9b). Quantitative analysis indicated that the amount of HR-NPs at tumor tissue is at least 4-fold higher than those in normal organs including liver, lung, spleen, kidney, and heart (Fig. 9c). The nanoparticular distribution at the hypoxic tumor tissue was also observed using the immunohistochemistry technique. As shown in Fig. 9d, the hypoxic tissue, stained by the FITC-labeled monoclonal

antibody, imbibed large amount of HR-NPs. These results suggest that HR-NPs can effectively reach the hypoxic tumor site after systemic administration in vivo. 3.5. In vivo anti-tumor efficacy of HR-NPs In an attempt to evaluate the anti-tumor efficacy, SCC7 tumorbearing mice were intravenously injected with saline, free DOX, or DOX-HR-NPs (Fig. 10a). The control group, treated with saline, exhibited a rapid increase in the tumor volume as a function of time. Free DOX also showed the considerable increase in the size, which is due to lack of tumor targetability. Notably, minimal increase in the tumor volume was found for the group treated with DOX-HR-NPs, suggesting the high anti-tumor efficacy. As expected, the mice treated with DOX-HR-NPs had the lowest weight among the groups (Fig. 10b). This high anti-tumor activity of DOX-HR-NPs might be due to their selective accumulation in tumor, followed by the intracellular release at the hypoxic cells (Fig. 8). 4. Conclusion In summary, we investigated the potential of hypoxiaresponsive nanoparticles as drug carriers. These carriers were stable in physiological conditions and capable of selectively releasing the hydrophobic drug under hypoxic conditions. DOX-loaded HRNPs showed higher toxicity to hypoxic cells than to normoxic cells. In addition, live animal NIRF imaging demonstrated that HR-NPs could effectively accumulate at the tumor site. As a consequence, DOX-HR-NPs exhibited enhanced anti-tumor efficacy, compared to free DOX. Overall, the results indicated that HR-NPs are promising drug carriers for selective delivery of hydrophobic drugs into hypoxic cells. Acknowledgments This work was financially supported by the Converging Research Program (20090081876) and the Basic Science Research Programs (20100027955 & 2012012827) of the NRF.

T. Thambi et al. / Biomaterials 35 (2014) 1735e1743

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Hypoxia-responsive polymeric nanoparticles for tumor-targeted drug delivery.

Hypoxia is a condition found in various intractable diseases. Here, we report self-assembled nanoparticles which can selectively release the hydrophob...
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