PEGylated Oxidized Alginate-DOX Prodrug Conjugate Nanoparticles

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PEGylated oxidized alginate-DOX prodrug conjugate nanoparticles crosslinked with fluorescent carbon dots for tumor theranostics Xu Jia, Mingliang Pei, Xubo Zhao, Kun Tian, Ting-Ting Zhou, and Peng Liu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00443 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 4, 2016

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ACS Biomaterials Science & Engineering

PEGylated oxidized alginate-DOX prodrug conjugate nanoparticles crosslinked with fluorescent carbon dots for tumor theranostics Xu Jia, Mingliang Pei, Xubo Zhao, Kun Tian, Tingting Zhou, and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China

AUTHOR INFORMATION *Corresponding Author. E-mail: [email protected]. Tel./Fax: 86-0931-8912582.

ACS Paragon Plus Environment

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Abstract Theranostic nanomedicine has recently emerged as an appealing approach for tumor chemotherapy. Here, for the first time, fluorescent carbon dots (Cdots) were used as crosslinker for tumor theranostic nanoparticles. Novel theranostic nanoparticles of approximately 26 nm with a doxorubicin (DOX) content of 0.2532 mg/mg (mPEG-OAL-DOX/Cdots) were designed by conjugating DOX onto mPEG-OAL/Cdots nanoparticles that were prepared by crosslinking the PEGylated oxidized alginate (mPEG-OAL) with Cdots. Due to the acid-labile Schiff base conjugating linkage, the theranostic prodrug nanoparticles showed low levels of DOX release in the simulated physiological media, which indicated premature drug leakage could be reduced during body circulation. Interestingly, the in vitro DOX release was triggered in the simulated tumor microenvironment without burst release. MTT assay and CLSM analysis showed that the mPEG-OAL/Cdots nanoparticles were non-cytotoxic, but that the mPEG-OAL-DOX/Cdots theranostic nanoparticles exhibited high degrees of inhibition of cancer cells due to their nucleustargeting DOX delivery. In, addition, their cellular fluorescence characteristic demonstrated a potential application for imaging-guided drug delivery in tumor treatment.

Keywords: tumor theranostics; prodrug nanoparticle; carbon dots; oxidized alginate; imagingguided drug delivery


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INTRODUCTION In recent years, theranostic nanomedicine, defined as an integrated ‘all-in-one’ nanotherapeutic platform for the simultaneous diagnosis, drug delivery and real-time tracking of chemotherapy efficiency,1 has become an appealing approach for tumor chemotherapy with advantages such as improved biodistribution, selective cancer targeting ability, reduced toxicity, masked drug efficacy, and minimum side effects.2 This platform has attracted increased interest for personalized nanomedicine, and follows the mantra of “the right drug for the right patient at the right moment”.3 To date, various tumor theranostic nanomedicines have been developed4 by incorporating imaging materials into common therapeutic strategies such as drug delivery, gene delivery, photodynamic therapy, hyperthermia, and radiation therapy.5 Due to their abundant functional groups, polymer nanoparticles including micelles can easily combine with most existing diagnostic and therapeutic agents. For chemotherapy, chemotherapeutic drugs could be conjugated onto polymer nanoparticles via acid-labile or reductant-cleavable linkages as prodrugs to achieve tumor-targeted, on-demand drug delivery to the tumor microenvironment.6 This occurs through the enhanced permeability and retention (EPR) effect.7 Therefore, the side effects and multiple drug resistance (MDR) of chemotherapeutic drugs in normal tissues could be minimized efficiently. Various imaging agents have been incorporated for diagnosis, such as gold nanoparticles,8 magnetic nanoparticles,9 fluorescent dyes,10 and contrast agents for magnetic resonance imaging (MRI),11 and so on. Fluorescent carbon dots (Cdots) are novel fluorescent carbon nanoparticles (less than 10 nm in size) with unique properties, such as specific quantum confinement effects, biocompatibility (specificially overcoming the high toxicity of traditional nanomaterials) and highly tunable photoluminescence (PL) properties. Generally, there are plentiful functional groups (e.g., –OH, -


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COOH and/or -NH2) on nanoparticle surfaces,12 thus endowing the Cdots with water solubility and a reaction ability. Moreover, their preparation procedure is simple. Therefore, fluorescent Cdots show great potential in photocatalysis, biochemical sensing, bioimaging, drug delivery, and other applications.13 Most recently, fluorescent Cdots have been used for thetumor theranostics as an excellent confocal and TPF imaging contrast agent and fluorescent pH-sensing probe. For example, Lv et al synthesized novel multifunctional GdOF:Ln@SiO2 (Ln = 10%Yb/1%Er/4%Mn) mesoporous capsules with strong up-conversion luminescent (UCL) GdOF:Ln as the core and a mesoporous silica layer as the shell, followed by modification with Cdots for multimodal imaging guided multiple therapies.14 Wang et al designed biocompatible hybrid nanogels by immobilizing fluorescent Cdots into biocompatible temperature/pH dual-responsive polymer nanogels via hydrogen bonding or complexation for simultaneous fluorescent pH sensing, TPF imaging, and NIR light/pH dual-responsive drug delivery.15 In the present work, fluorescent Cdots containing plentiful surface amino groups were used for the first time as a crosslinker for tumor theranostics. Alginate, a natural polysaccharide, could be easily crosslinked with amino-functionalized nanoparticles for controlled antitumor drugdelivery.16 After crosslinking the PEGylated oxidized alginate (mPEG-ALG) with the fluorescent Cdots, the prodrug conjugate theranostic nanoparticles (mPEG-OAL-DOX/Cdots), were prepared by conjugating doxorubicin (DOX) onto the mPEG-OAL/Cdots nanoparticles via the acid-labile Schiff base linkage, which acted as an “off-on” switch for the pH-triggered release of DOX in the acidic tumor microenvironment (Scheme 1).


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Scheme 1. Outline for the synthesis of the theranostic prodrug nanoparticles.

EXPERIMENTAL SECTION

Materials. Methoxypolyethylene glycol amine (mPEG-NH2, Mn = 2000 Da, 99%) was acquired from Beijing Kaizheng Biotech. Development Co., Ltd. (Beijing, China). Sodium alginate (AL, chemical pure grade, viscosity of 30-80 cP for 1.0 mg/mL solution and viscosity-average molecular weight of 4.5 × 105) was obtained from Xudong Chemical Plant (Beijing, China). Citric acid anhydrous (CA, 99.5%) was bought from Sinopharm Chemical Reagent Co., Ltd. Ethylenediamine (EDA, 99%) was purchased from Rionlon Bohua (Tianjin) Pharmaceutical


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Chemical Co. Ltd. (Tianjin, China). Sodium periodate (99.5%) was obtained from Zhejiang Hichi Chemical Co., Ltd. (Yuhuan, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

(EDC⋅HCl,

99%)

was

provided

by

J&K

Chemical

Co.,

Ltd.

N-

Hydroxysuccinimide (NHS, 98%) was purchased from Aladdin Chemistry Co., Ltd. Doxorubicin hydrochloride (DOX⋅HCl, 99.4%) was provided by Beijing Huafeng United Technology Co., Ltd. (Beijing, China). Double-deionized water was used throughout the experiment.

Carbon dots (Cdots). The Cdots containing abundant amino groups were prepared via a onestep microwave pyrolysis as reported previously,17 with a yield of 49.4%. Briefly, 2.0 g (10.41 mmol) of CA was dissolved in 20 mL of water in a 100 mL beaker, and 1.04 mL (15.62 mmol) of EDA was added under vigorous stirring to form a clear and transparent solution. The beaker was subsequently placed at the center of the rotation plate of a domestic microwave oven (700 W) and heated for 4 min until the solution changed from colorless to red-brown. After cooling to room temperature, the Cdots were washed by dialysis (MWCO of 1000) with water for 3 days, and separated by lyophilization.

Oxidized alginate (OAL). Sodium alginate (3.0 g) was dissolved in 300 mL of water under magnetic stirring, and 50 mL of sodium periodate (2.0 g, 9.35 mmol) aqueous solution was added. The reaction mixture was stirred in the dark for 24 h. Then the product was precipitated by adding 400 mL of ethanol. The precipitant was centrifuged, washed with a mixture of water and ethanol (v/v, 1:1), and dried under vacuum at room temperature for 48 h.18 The degree of oxidation (defined as the number of the oxidized guluronate units per 100 guluronate units) was measured to be 27.8% with iodometry.18


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PEGylated oxidized alginate (mPEG-OAL). First, 1.0 g of OAL was dissolved in 100 mL of water. Then, EDC⋅HCl (0.8870 g, 4.63 mmol) and NHS (0.5325 g, 4.63 mmol) were added to the solution and the pH was adjusted to 4.5. After the carboxyl groups had been activated for 4 h at room temperature, mPEG-NH2 (1.0 g, 0.5 mmol) was added to the reaction under magnetic stirring, and the reaction was conducted for 48 h at room temperature. Finally, the reaction solution was dialyzed (MWCO of 14000) against water for 3 days and the product was gathered by lyophilization.19 Nitrogen elemental analysis showed that 8.27% of the carboxyl groups in OAL were amidated with mPEG-NH2.

mPEG-OAL/Cdots nanoparticles. First, 1.5 g of mPEG-OAL was dissolved in 100 mL of water. Then, EDC⋅HCl (0.8870 g, 4.63 mmol) and NHS (0.5325 g, 4.63 mmol) were added into the above-mentioned solution and the pH was adjusted to 4.5. After pre-activating the carboxyl groups at room temperature for 4 h, 0.075 g of Cdots was added into the reaction solution under magnetic stirring, and the mixture was stirred for 48 h to allow the crosslinking of the mPEGOAL with Cdots via amidation. Finally, the reaction solution was handled with dialysis (MWCO of 14000) for 3 days and the resultant mPEG-OAL/Cdots nanoparticles were gathered by lyophilization.20 The Cdots mass content in the mPEG-OAL/Cdots nanoparticles was determined to be 2.48% by measuring the fluorescence intensity at the excitation wavelength of 350 nm.

Cellular fluorescence imaging. The cellular fluorescence imaging function of the mPEG-OALDOX/Cdots nanoparticles was evaluated with confocal laser scanning microscope (CLSM). BHK cells were cultured in a nutrient solution containing 10% FBS (fetal bovine serum). The cells were maintained under a humidified atmosphere containing 5% CO2 at 37 °C for 12 h. After removing the cultured solution, the cells were incubated in the presence of 100 µg/mL mPEG-


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OAL/Cdots in 1.0 mL of fresh culture medium for 2 h. Finally the cells were washed three times with PBS to remove the residual nanoparticles before the imaging measurements.

mPEG-OAL-DOX/Cdots theranostic prodrug nanoparticles. First, 0.30 g of mPEGOAL/Cdots nanoparticles was dispersed in 24 mL of a water and ethanol mixture (v/v, 5:3). After 2 drops of glacial acetic acid was added, 15 mL of DOX⋅HCl (0.1599 g, 0.29 mmol) aqueous solution was injected. The mixture was reacted at 60 °C for 48 h under nitrogen atmosphere in the dark. Finally, the mPEG-OAL-DOX/Cdots nanoparticles were collected by centrifugation and washed with a mixture of water and ethanol (v/v, 3:1) several times until the supernatant was colorless to ensure the complete removal of the adsorbed DOX via non-covalent bonds such as electrostatic interaction or hydrogen binding. The product was dried under vacuum at room temperature and stored at 4 °C in the dark.

DOX content in theranostic nanoparticles and in vitro release performance. To determine the DOX content in the theranostic nanoparticles, 4.5 mg of mPEG-OAL-DOX/Cdots was dispersed into 20 mL of 1 M HCl aqueous solution at room temperature for 48 h.21 The DOX concentration was determined using a UV-vis spectrometer at 480 nm. The DOX content of the mPEG-OAL-DOX/Cdots nanoparticles was calculated to be 0.2532 mg/mg, which is the mass ratio of DOX and the mPEG-OAL-DOX/Cdots nanoparticles. The in vitro release of DOX from the mPEG-OAL-DOX/Cdots nanoparticles was evaluated at 37 °C in three different buffer solutions: pH 5.0 acetate buffer solution (ABS), pH 6.5 ABS and pH 7.4 phosphate buffer solution (PBS). Then, 10.0 mg of the mPEG-OAL-DOX/Cdots nanoparticles was dispersed in 10 mL of buffer solution, and the dispersion was transferred into a dialysis bag with a molecular weight cut off of 14000. The dialysis bag was then immersed into


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120 mL of the corresponding buffer solution and mildly shaken at 37 °C. After certain time intervals, 5 mL of the solution was removed to measure the concentration of the released DOX with a UV−vis spectrometer at 480 nm. To keep the solution volume constant, an equal volume of the fresh corresponding buffer solution was replenished after each sampling.

Cytotoxicity and cellular uptake. The cytocompatibility of the mPEG-OAL/Cdots and mPEGOAL-DOX/Cdots nanoparticles was evaluated in SKOV3 cells by an MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. SKOV3 cells were implanted into a 96-well plate at a density of 1 ×105 cells/well. Then, mPEG-OAL/Cdots and mPEG-OALDOX/Cdots nanoparticles were added onto the cells at differing concentrations, and the cells were incubated at 37 °C for 24 h. Next, 20 µL of a mg/mL MTT solution was injected into each well. Four hours later, the liquid was removed, and 150 µL of DMSO was added to each well to dissolve the crystal substance for 20 min. Finally, the cell viability was detected by measuring the absorbance at a wavelength of 490 nm using an Enzyme-linked Immunosorbent Assay Appliance. Cellular uptake was characterized with a CLSM technique in the SKOV3 cells. The cells were cultivated in a 24-well plate (1 × 105 cells/well) with 100 µL of culture solution for 12 h at 37 °C. Then, 100 µL of culture solution containing 40 µg/mL of the mPEG-OAL-DOX/Cdots nanoparticles was added to the microplates, and the cells were incubated for another 12 h. Then, the nutrient solution was discarded and the cells were washed with culture solution to remove the prodrug residue. Finally, the cell nuclei were dyed with Hoechst 33258 and fixed with 4% paraformaldehyde. The location of intracellular fluorescence was traced at the excitation wavelengths of 480 nm for DOX and 405 nm for Hoechst 33258.


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Instruments and characterization. The 1H NMR spectra of the samples were characterized using a Nuclear Magnetic Resonance instrument (JEOL ECS 400M) at 400 MHz with deuterium oxide as a solvent. The Fourier transform infrared (FT-IR) spectra were recorded with a Bruker IFS 66 v/s infrared spectrometer in 400−4000 cm−1 with a resolution of 4 cm−1, using the potassium bromide (KBr) pellet technique. Nitrogen elemental analysis was performed on an Elemental Analyzer vario EL Cube instrument. The morphology and size of the Cdots and nanoparticles were characterized by JEM-1200EX transmission electron microscopy (TEM), with their aqueous dispersions. The hydrodynamic diameter and distribution of the products were determined with their aqueous dispersions on a dynamic light scattering instrument (DLS, BI-200SM) at ambient temperature. The steady-state emission spectra of the Cdots and nanoparticles were detected with their aqueous dispersions using a Hitachi F-7500 fluorescence spectrometer. The DOX content in the theranostic nanoparticles (mPEG-OAL-DOX/Cdots) and their cumulative release were measured with a UV-vis spectrometer (TU-1901) at a wavelength of 480 nm at room temperature.

RESULTS AND DISCUSSION

Preparation and characterization of mPEG-OAL/Cdots nanoparticles. The fluorescent Cdots containing plentiful amino groups were prepared through a microwave pyrolysis reaction


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of citric acid and ethylenediamine.17 The near-spherical Cdots were obtained with a particle size of approximately 2.5 nm, as measured by TEM analysis (Figure 1a). Due to the hydrophilic groups on their surface, they possessed a high water swelling capacity, with an average hydrodynamic diameter (Dh) of approximately 4.5 nm from the DLS analysis (Figure 2a).

Figure 1. TEM images of the Cdots (a) and mPEG-OAL-DOX/Cdots nanoparticles (b).

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The PEGylated oxidized alginate/carbon dots (mPEG-OAL/Cdots) nanoparticles were then prepared by amidation between the carboxyl groups of the mPEG-OAL and the amino groups of the Cdots (Scheme 1). The 1H NMR spectrum of the mPEG-OAL showed the chemical shifts of PEG at δ = 3.64 ppm and δ = 3.38 ppm and the chemical shifts of OAL ranged from 3.70 ppm to 4.50 ppm (Figure 3). In addition, the chemical shifts of both the mPEG-OAL (in 3.3-4.5 ppm) and the Cdots (1.13 ppm) could be seen in the 1H NMR spectrum of the mPEG-OAL/Cdots nanoparticles, thus revealing the successful preparation of the functional mPEG-OAL/Cdots nanoparticles. These reactions were also revealed by FT-IR analysis (Figure 4). The stretching vibrations of the -CH2- and C-O groups in PEG appeared at 2873 cm-1 and 1113 cm-1 in the infrared spectrum of mPEG-OAL. After crosslinking with Cdots, the N–H stretch of the residual amino groups in the Cdots appeared in the infrared spectrum of the mPEG-OAL/Cdots nanoparticles.


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Figure 3. 1 H NMR spectra of Cdots (a), OAL (b), mPEG-OAL (c) and mPEG-OAL/Cdots (d).

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mPEG-OAL/Cdots

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Figure 4. FT-IR spectra of Cdots, OAL, mPEG-OAL, mPEG-OAL/Cdots and mPEG-OALDOX/Cdots.


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Figure 5. PL emission spectra of Cdots (0.1 mg/mL) (a) and mPEG-OAL/Cdots (2.5 mg/mL) (b) aqueous dispersions and normalized PL emission spectra of the aqueous dispersions of Cdots (c) and mPEG-OAL/Cdots (d) under different wavelength excitations, and PL intensity Insets: Fluorescence images of Cdots and mPEG-OAL/Cdots aqueous dispersions under UV light (365 nm).

The fluorescence emission spectra of the Cdots (Figure 5a) and mPEG-OAL/Cdots (Figure 5b) were characterized under different excitation wavelengths. From the normalized fluorescence


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emission spectra of both the Cdots (Figure 5c) and mPEG-OAL/Cdots (Figure 5d), it was easily observed that the emission wavelength transformed into the long-wavelength band by increasing the excitation wavelength, because the emission trap on their surface was distributive.22 Although the Cdots were conjugated onto the mPEG-OAL through amidation, their fluorescence characteristic was still maintained, which makes them potentially useful for imaging-guided drug delivery in tumor treatment. After incubation with the mPEG-OAL/Cdots nanoparticles (100 µg/mL) for 12 h, BHK cells showed blue luminescence following excitation at 365 nm (Figure 6). The results indicated that mPEG-OAL/Cdots nanoparticles could be taken up by BHK cells via endocytosis and then imaged within the cells.

Figure 6. Cellular uptake of BHK cells after incubation with the mPEG-OAL/Cdots nanoparticles (100 µg/mL) for 12 h by CLSM technique. From left to right, bright-field image (a) and confocal fluorescence image (b).

Preparation and characterization of mPEG-OAL-DOX/Cdots theranostic prodrug nanoparticles. Due to its reversibility, the acid-labile Schiff base linkage has been widely implemented in pH-triggered drug delivery systems.23-25 Here, DOX was conjugated onto the


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mPEG-OAL/Cdots nanoparticles via the acid-labile Schiff base linkage. The theranostic nanoparticles (mPEG-OAL-DOX/Cdots) were spherical, with a particle size of 27.3±2.86 nm (Figure 1b). After swelling, their average hydrodynamic diameter was 55.5±1.47 nm (Figure 2b). Such size is expected to enhance treatment efficiency via the EPR effect.7 A new absorbance of the Schiff base bonds appeared at 1715 cm-1 in the FT-IR spectrum of the product (Figure 2),26 which indicated that the mPEG-OAL-DOX/Cdots theranostic nanoparticles were successfully prepared. The DOX content in the theranostic nanoparticles was determined to be 0.2532 mg/mg by UV-vis measurement at 480 nm, after destroying the Schiff base linkage in 1 M HCl for 48 h.21

pH-triggered controlled release. To evaluate the controlled release performance, mPEG-OALDOX/Cdots nanoparticles were dispersed into three buffer solutions (pH 5.0, pH 6.5, and pH 7.4) at 37 °C, and the controlled release profiles were illustrated in Figure 7. We observed that DOX release increased with the increase of the media acidity. That is, the drug release was triggered by the media acidity, namely the pH triggering effect, due to the acid-labile Schiff base linkage for the conjugation of DOX onto the nanoparticles.26 The cumulative release ratio was 57.0% in the pH 5.0 buffer solution within 60 h, whereas it was 32.4% in the pH 6.5 buffer solution. In contrast, the Schiff base was relatively stable in the simulated physiological medium with a cumulative release ratio of only 13.0% over the same releasing time, thus demonstrating the potential suppressed drug leakage during body circulation. The in vitro controlled release indicated that the theranostic nanoparticles could realize site-specific and on-demand release targeted to tumor tissues.


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Figure 8. The profiles of drug release mechanism based on the Higuchi and Korsmeyer-Peppas models.

The release profiles in the different buffer solutions were fitted with the Higuchi and Korsmeyer-Peppas equation (Figure 8). For both models, the linear coefficients were relatively ideal, with R2 > 0.94. The Higuchi model demonstrated that the release of DOX was diffusioncontrolled in the pH 5.0 solution (the slope > 1), but in the other solutions, there was nondiffusion controlled release (the slope < 1).27,28 Furthermore, the release in the three media was non-Fickian diffusion according to the Korsmeyer-Peppas model, because their slopes were higher than 0.5.29

In vitro cytotoxicity and cellular uptake. The cytocompatibility of the mPEG-OAL/Cdots and mPEG-OAL-DOX/Cdots nanoparticles were investigated using MTT assays in SKOV3 cells at different concentrations (5, 10, 20, and 40 µg/mL) after 24 h of incubation. The cell viability ranged from 103% to 98.4% with increasing concentrations of the mPEG-OAL/Cdots (Figure 9), thus indicating their cytocompatibility. However, the cell viability decreased significantly after injection of mPEG-OAL-DOX/Cdots nanoparticles; only 45.5% of the cells survived in the


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culture solution with a prodrug concentration of 20 µg/mL. The antitumor activity of the mPEGOAL-DOX/Cdots prodrug was also assessed by a series of controlled tests with free DOX. Because the DOX content was 0.2532 mg/mg in the prodrug and the sustained DOX release had a release ratio of 47.2% within 24 h, the actual cumulated DOX concentration within 24 h in the case of the mPEG-OAL-DOX/Cdots was only 12% that of the scenario with free DOX. Surprisingly, the former showed approximately half of the SKOV3 cell killing rate compared to free DOX, with a concentration of no more than 10 µg/mL, although the 12% DOX (1/8 dosage of free DOX) was dosed in a manner of sustained release. These results indicated that the enhanced anticancer efficacy of DOX could be introduced in the form of the proposed prodrug nanoparticles. Therefore, we conclude that the prodrug nanoparticles possess potential oncotherapeutic applications due to their high inhibition of cancer cells.

mPEG-OAL/Cdots mPEG-OAL-DOX/Cdots Free DOX

120 100

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20 0 0

10

20

30

40

50

Concentration (µg/mL)

Figure 9. Cell viability of the SKOV3 cells in the MTT assay after incubating with mPEGOAL/Cdots, mPEG-OAL-DOX/Cdots and free DOX for 24 h. Data are presented as the mean ± standard deviation (SD; n=6).


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The cellular uptake of the mPEG-OAL-DOX/Cdots nanoparticles was explored in SKOV3 cells using the CLSM technique after 24 h of incubation. Blue fluorescence showed the position of the cell nuclei stained with Hoechst 33258 (Figure 10a), whereas red fluorescence showed the released DOX from the prodrug nanoparticles (Figure 10b). In the merged image (Figure 10c), the fluorescence positions were completely overlapping, demonstrating that the released DOX was mainly accumulated in the cell nuclei. These results indicated that the prodrug nanoparticles enabled nucleus-targeted DOX delivery,30 which induced DNA damage and cytotoxicity in the cells31.

Figure 10. Cellular uptake of SKOV3 cells stained by Hoechst 33258 and incubated with the mPEG-OAL-DOX/Cdots prodrug (20 µg/mL) for 12 h by CLSM technique. From left to right, cell nuclei images are stained by Hoechst 33258 (a), DOX fluorescence in cells (b) and merged images (c).

CONCLUSIONS

In summary, for the first time, the fluorescent carbon dots (Cdots) containing plentiful amino groups of approximately 2.5 nm were used as a crosslinker for PEGylated oxidized alginate


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(mPEG-OAL) to design natural polysaccharide-based theranostic nanoparticles. As a result, novel mPEG-OAL-DOX/Cdots nanoparticles were obtained with a particle size of 26 nm and DOX content of 0.2532 mg/mg, after conjugating DOX via the acid-labile Schiff base linkage. The incorporation of fluorescent Cdots enabled imaging-guided drug delivery in the tumor therapy. Furthermore, the pH-triggered site-specific release and tumor-targeted delivery of DOX from the prodrug nanoparticles in the mimicked tumor microenvironment enhanced the anticancer efficiency of DOX, with less drug leakage in the simulated physiological media. This system provides a promising strategy for advanced anticancer theranostic applications.

AUTHOR INFORMATION Corresponding Author * Corresponding Author. Tel./Fax: 86 0931 8912582. Email: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This project was granted financial support from the National Natural Science Foundation of China (Grant no. 20904017), and the Program for New Century Excellent Talents in University (Grant no. NCET-09-0441).

REFERENCES


22

Page 23 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(1) Lammers, T.; Kiessling, F.; Hennink, W. E.; Strom, G. Nanotheranostics and Image-Guided Drug Delivery: Current Concepts and Future Directions. Mol. Pharmaceutics 2010, 7, 1899– 1912 DOI: 10.1021/mp100228v. (2) Kumar, R.; Shin, W. S.; Sunwoo, K.; Kim, W. Y.; Koo, S.; Bhuniya, S.; Kim, J. S. Small Conjugate-based Theranostic Agents. Chem. Soc. Rev. 2015, 44, 6670–6683 DOI: 10.1039/C5CS00224A. (3) Fan, Z.; Fu, P. P.; Yu, H. T.; Ray, P. C. ,Theranostic Nanomedicine for Cancer Detection and Treatment. J. Food Drug Anal. 2014, 22, 3–17 DOI: 10.1016/j.jfda.2014.01.001. (4) Skeete, Z.; Cheng, H. W.; Crew, E.; Lin, L. Q.; Zhao, W.; Joseph, P.; Shan, S. Y.; Cronk, H.; Luo, J.; Li, Y. J.; Zhang, Q. W.; Zhong, C. J. Design of Functional Nanoparticles and Assemblies for Theranostic Applications. ACS Appl. Mater. Interface 2014, 6, 21752–21768 DOI: 10.1021/am502693t. (5) Luk, B. T.; Zhang, L. F. Current Advances in Polymer-Based Nanotheranostics for Cancer Treatment and Diagnosis. ACS Appl. Mater. Interface 2014, 6, 21859–21873 DOI: 10.1021/am5036225. (6) Shen, Y. Q.; Ed.; Functional Polymers for Nanomedicine, Chapter 11, Royalty Society of Chemistry Publishing, 2013. (7) Maeda, H.; Nakamura, H.; Fang, J. The EPR Effect for Macromolecular Drug Delivery to Solid Tumors: Improvement of Tumor Uptake, Lowering of Systemic Toxicity, and Distinct Tumor

Imaging

In

Vivo.

Adv.

Drug

Deliv.

Rev.

2013,

65,

71–79

DOI:

10.1016/j.addr.2012.10.002. (8) Dixit, S.; Miller, K.; Zhu, Y.; McKinnon, E.; Novak, T.; Kenney, M. E.; Broome, A.-M. Targeting Wnt Canonical Signaling by Recombinant sFRP1 Bound Luminescent Au-


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

Nanocluster Embedded Nanoparticles in Cancer Theranostics. ACS Biomater. Sci. Eng. 2015, 1, 1256–1266 DOI: 10.1021/acsbiomaterials.5b00305. (9) Jaiswal, M.; De, M.; Chou, S. S.; Vasavada, S.; Bleher, R.; Prasad, P. V.; Bahadur, D.; Dravid, V. P. Thermoresponsive Magnetic Hydrogels as Theranostic Nanoconstructs. ACS Appl. Mater. Interface 2014, 6, 6237–6247 DOI: 10.1021/am501067j. (10) Wang, G. S.; Ma, Y. Y.; Wei, Z. Y.; Qi, M. Development of Multifunctional Cobalt Ferrite/Graphene Ooxide Nanocomposites for Magnetic Resonance Imaging and Controlled Drug Delivery. Chem. Eng. J. 2016, 289, 150–160 DOI: 10.1016/j.cej.2015.12.072. (11) Fu, L. Y.; Sun, C. Y.; Yan, L. F. Galactose Targeted pH-Responsive Copolymer Conjugated with Near Infrared Fluorescence Probe for Imaging of Intelligent Drug Delivery. ACS Appl.Mater. Interface 2015, 7, 2104–2115 DOI: 10.1021/am508291k. (12) Cao, L.; Wang, X.; Meziani, M. J.; Lu, F. S.; Wang, H. F.; Luo, P. G.; Lin, Y.; Harruff, B. A.; Veca, L. M.; Murray, D.; Xie, S. Y.; Sun, Y. P. Carbon Dots for Multiphoton Bioimaging. J. Am. Chem. Soc. 2007, 129, 11318–11319 DOI: 10.1021/ja073527l. (13) Wang, Y. F.; Hu, A. G. Carbon Quantum Dots: Synthesis, Properties and Applications. J. Mater. Chem. C 2014, 2, 6921–6939 DOI: 10.1039/C4TC00988F. (14) Lv, R. C.; Yang, P. P.; He, F.; Gai, S. L.; Li, C. X.; Dai, Y. L.; Yang, G. X.; Lin, J. A Yolklike Multifunctional Platform for Multimodal Imaging and Synergistic Therapy Triggered by a Single Near-Infrared Light. ACS Nano 2015, 9, 1630–1647 DOI: 10.1021/nn5063613. (15) Wang, H.; Di, J.; Sun, Y. B.; Fu, J. P.; Wei, Z. Y.; Matsui, H.; Alonso, A. C.; Zhou, S. Q. Biocompatible PEG-Chitosan@Carbon Dots Hybrid Nanogels for Two-Photon Fluorescence Imaging, Near-Infrared Light/pH Dual-Responsive Drug Carrier, and Synergistic Therapy. Adv. Funct. Mater. 2015, 25, 5537–5547 DOI: 10.1002/adfm.201501524.


24

Page 25 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(16) Barbucci, R.; Giani, G.; Fedi, S.; Bottari, S.; Casolaro, M. Biohydrogels with Magnetic Nanoparticles as Crosslinker: Characteristics and Potential Use for Controlled Antitumor Drug Delivery. Acta Biomater. 2012, 8, 4244–4252 DOI: 10.1016/j.actbio.2012.09.006. (17) Cheng, L.; Li, Y. M.; Zhai, X. Y.; Xu, B.; Cao, Z. Q.; Liu, W. G. Polycation-bpolyzwitterion Copolymer Grafted Luminescent Carbon Dots as a Multifunctional Platform for Serum-Resistant Gene Delivery and Bioimaging. ACS Appl. Mater. Interface 2014, 6, 20487–20497 DOI: 10.1021/am506076r. (18) Mu, B.; Lu, C. Y.; Liu, P. Disintegration-controllable Stimuli-Responsive Polyelectrolyte Multilayer Microcapsules via Covalent Layer-by-Layer Assembly. Colloid Surf. BBiointerface 2011, 82, 385–390 DOI: 10.1016/j.colsurfb.2010.09.024. (19) Zhao, X. B.; Liu, L.; Li, X. R.; Zeng, J.; Jia, X.; Liu, P. Biocompatible Graphene Oxide Nanoparticle-based Drug Delivery Platform for Tumor Microenvironment-Responsive Triggered

Release

of

Doxorubicin.

Langmuir

2014,

30,

10419–10429

DOI:

10.1021/la502952f. (20) Goh, E. J.; Kim, K. S.; Kim, Y. R.; Jung, H. S.; Beack, S.; Kong, W. H.; Scarcelli, G.; Yun, S. H.; Hahn, S. K. Bioimaging of Hyaluronic Acid Derivatives Using Nanosized Carbon Dots. Biomacromolecules 2012, 13, 2554–2561 DOI: 10.1021/bm300796q. (21) Xu, X. W.; Floresand, J. D.; McCormick, C. L. Reversible Imine Shell Cross-Linked Micelles from Aqueous RAFT-Synthesized Thermoresponsive Triblock Copolymers as Potential Nanocarriers for “pH-Triggered” Drug Release. Macromolecules 2011, 44, 1327– 1334 DOI: 10.1021/ma102804h. (22) Zhu, H.; Wang, X. L.; Li, Y. L.; Wang, Z. J.; Yang, F.; Yang, X. R. Microwave Synthesis of Fluorescent Carbon Nanoparticles with Electrochemiluminescence Properties. Chem. Commun. 2009, 34, 5118–5120 DOI: 10.1039/B907612C.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

(23) Cui, J.; Yan, Y.; Wang, Y.; Caruso, F. Templated Assembly of pH-Labile Polymer-Drug Particles for Intracellular Drug Delivery. Adv. Funct. Mater. 2012, 22, 4718-4723 DOI: 10.1002/adfm.201201191. (24) Guo, X.; Shi, C.; Wang, J.; Di, S.; Zhou, S. pH-Triggered Intracellular Release from Actively Targeting Polymer Micelles. Biomaterials 2013, 34, 4544-4554 DOI: 10.1016/j.biomaterials.2013.02.071 (25) Li, F.; He, J,; Ni, P. A pH-Sensitive and Biodegradable Supramolecular Hydrogel Constructed from a PEGylated Polyphosphoester-Doxorubicin Prodrug and α-Cyclodextrin Polym. Chem., 2015, 6, 5009-5014 DOI: 10.1039/c5py00620a (26) Xiao, N. Y.; Liang, H.; Lu, J. Degradable and Biocompatible Aldehyde-functionalized Glycopolymer Conjugated with Doxorubicinvia Acid-labile Schiff Base Linkage for pHtriggered Drug Release. Soft Matter 2011, 7, 10834–10840 DOI: 10.1039/C1SM06181J. (27) Siepmann, J.; Peppas, N. A. Higuchi Equation: Derivation, Applications, Use and Misuse. Int. J. Pharm. 2011, 418, 6–12 DOI: 10.1016/j.ijpharm.2011.03.051. (28) Kalia, Y. N.; Guy, R. H. Modeling Transdermal Drug Release. Adv. Drug Deliv. Rev. 2001, 48, 159–172 DOI: 10.1016/S0169-409X(01)00113-2. (29) Costa, P.; Lobo, J. M. S. Modeling and Comparison of Dissolution Profiles. Eur. J. Pharm. Sci. 2001, 13, 123–133 DOI: 10.1016/S0928-0987(01)00095-1. (30) Guo, H; Yang, C; Hu, Z; Wang, W; Wu, Y; Lai, Q; Yuan, Z. Ethylene Glycol Oligomer Modified-Sodium Alginate for Efficiently Improving the Drug Loading and the Tumor Therapeutic Effect. J. Mater. Chem. B 2013, 1, 5933–5941 DOI: 10.1039/c3tb20968g. (31) Frederick C. A; Williams L. D, Ughetto G; Van der Marel, G. A.; Van Boom, J. H.; Rich, A.; Wang, A. H. J. Structural Comparison of Anticancer Drug-DNA Complexes: Adriamycin

and

Daunomycin.

Biochemistry

1990,


29,

2538–2549

DOI:

26

Page 27 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10.1021/bi00462a016.


27


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Graphical Abstract


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PEGylated Oxidized Alginate-DOX Prodrug Conjugate Nanoparticles

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