Dual-Responsive Carbon Dots for Tumor Extracellular

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Dual-Responsive Carbon Dots for Tumor Extracellular Microenvironment Triggered Targeting and Enhanced Anticancer Drug Delivery Tao Feng, Xiangzhao Ai, Huimin Ong, and Yanli Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06695 • Publication Date (Web): 01 Jul 2016 Downloaded from http://pubs.acs.org on July 2, 2016

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ACS Applied Materials & Interfaces

Dual-Responsive Carbon Dots for Tumor Extracellular Microenvironment Triggered Targeting and Enhanced Anticancer Drug Delivery Tao Feng,† Xiangzhao Ai,† Huimin Ong,† Yanli Zhao*,†,‡ †

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical

Sciences, Nanyang Technological University, 21 Nanyang Link, 637371, Singapore ‡

School of Materials Science and Engineering, Nanyang Technological University, 50

Nanyang Avenue, 639798, Singapore

ABSTRACT: In this work, pH/redox dual-responsive carbon dots (CDs-RGD-Pt(IV)-PEG) were fabricated for tumor extracellular microenvironment triggered targeting and enhanced anticancer drug delivery. The system consists of fluorescent carbon dots as imaging-guided drug nanocarriers, cisplatin(IV) as prodrug, and RGD peptide as active targeting ligand, which is covered by monomethoxypolyethylene glycol (mPEG) through tumor extracellular pH (6.5-6.8) responsive benzoic-imine bond. The drug nanocarriers could be tracked by multicolor fluorescence of carbon dots. After the hydrolysis of benzoic-imine bond at the tumor extracellular pH to expose the inner targeting RGD peptide, the drug nanocarriers showed effective uptake by cancer cells through RGD-integrin αvβ3 (ligand-receptor) interaction. Upon the internalization, the loaded cisplatin(IV) prodrug was reduced to cytotoxic cisplatin in reductive cytosol of cancer cells to exhibit therapeutic effects. Confocal imaging, flow cytometry and cell viability assays using CDs-RGD-Pt(IV)-PEG were carried out to reveal the enhanced uptake and better therapeutic efficiency to cancer cells with high integrin αvβ3 expression at tumor extracellular pH than that in physiological condition. The

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developed CDs-RGD-Pt(IV)-PEG offers a new strategy to provide safe and effective therapeutic agents based on carbon dots for promising cancer therapy. KEYWORDS: carbon dots, dual responsiveness, imaging-guided delivery, tumor targeting, enhanced cancer therapy

INTRODUCTION In the past decades, imaging-guided cancer therapy is a growing field to pave the way for personalized cancer treatment.1-5 Among various imaging techniques such as positron emission tomography (PET), single photon emission computed tomography (SPECT), X-ray computed tomography (CT), ultrasound imaging (US) and magnetic resonance imaging (MRI), fluorescent imaging has the advantages of high specificity, high contrast and excellent sensitivity.6 Commonly used fluorescent imaging agents are organic dyes or semiconducting quantum dots (QDs), but they have the problems of photobleaching or innate toxicity.7-11 Carbon dots (CDs) have emerged as a new class of fluorescent imaging agents owing to their outstanding merits in terms of high water solubility, good photostability, excellent biocompatibility, easy surface functionalization and excitation-dependent emission.12-21 In addition to in vitro and in vivo fluorescent imaging of cancer cells and living animals, CDs were used as nanocarriers for fluorescent imaging-guided delivery of chemotherapy drugs, photosensitizers, and therapeutic genes.22-28 For a drug delivery system, active targeting can enhance cellular internalization of drug carriers by utilizing specific interactions between targeting ligands and corresponding receptors overexpressed on the surface of cancer cells, resulting in efficient cancer

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therapy.29-36 Commonly used targeting ligands are small molecules (e.g., folic acid), peptides (e.g., RGD (Arg-Gly-Asp) and TAT (Tyr-Gly-Arg-(Lys)2-(Arg)2-Gln-(Arg)3)), antibodies and aptamers.37 The targeting ligands are usually attached on the surface of drug carriers. Such approach has two main disadvantages.38,39 The first one is that reticular endothelial system can recognize the targeting ligands directly or through opsonins, enhancing early clearance of the carriers from blood circulation.38 The second one is that corresponding receptors to the targeting ligands are not only overexpressed by cancer cells, but also expressed on the surface of normal cells. Therefore, the drug carriers with targeting ligands can cause side effects to the normal cells.39 To solve the issues, tumor extracellular microenvironment (e.g., slightly acidic pH or overexpressed enzyme) triggered targeting strategy is proposed, in which targeting ligands are hidden inside the stealth layer of the carriers during the blood circulation. Upon the accumulation in the tumor tissue through the enhanced permeation and retention (EPR) effect, the targeting ligands become to present on the surface of drug carriers to facilitate the interaction with receptors on the surface of cancer cells.40-45 Although several active targeting drug delivery platforms utilizing fluorescent CDs have been developed,22,26 there is still no report on fabricating CD-based drug carriers with tumor extracellular microenvironment triggered targeting characteristics. Herein,

imaging-guided

and

pH/redox

dual-responsive

CD

carriers

(CDs-RGD-Pt(IV)-PEG) containing targeting ligand RGD peptide were developed (Scheme 1), which were PEGylated at normal physiological condition (pH 7.4). The RGD peptide could

be

exposed

at

tumor

extracellular

microenvironment

(pH

6.5-6.8).

CDs-RGD-Pt(IV)-PEG could be tracked by multicolor fluorescence of CDs, and the

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tumor-triggered targeting property was offered by the hydrolysis46-48 of benzoic-imine bond at pH 6.8 to deshield the RGD peptide, leading to effective ligand-receptor interaction and enhanced uptake by cancer cells overexpressing integrin αvβ3 on the cellular membrane. Upon the internalization, the loaded cisplatin(IV) prodrug in CDs-RGD-Pt(IV)-PEG was reduced to cisplatin in reductive cytosol of cancer cells to exhibit the cytotoxicity.49,50 The in vitro results indicate that CDs-RGD-Pt(IV)-PEG is a promising candidate in the cancer treatment.

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Scheme 1: (a) Schematic illustration for the preparation of CD-based drug nanocarrier CDs-RGD-Pt(IV)-PEG with tumor triggered targeting property. (b) Schematic illustration of the drug delivery process using CDs-RGD-Pt(IV)-PEG: (1) tracking drug nanocarriers by multicolor fluorescence of CDs with PEGylation in normal physiological condition, (2) tumor-triggered targeting ligands exposed at tumor extracellular pH 6.8, (3) effective uptake by cancer cells through ligand-receptor interaction, (4) cisplatin release from cisplatin(IV) prodrug under reductive cytosol, and (5) cisplatin binding with DNA to exhibit the cytotoxicity.

EXPERIMENTAL SECTION Materials. Chemicals including 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), citric acid, cis-[PtCl2(NH3)2], N,N′-dicyclohexylcarbodiimide (DCC),

diethylenetriamine,

N,N-diisopropylethylamine

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride

(DIPEA), (EDC•HCl),

4-dimethylaminopyridine (DMAP), 4-formylbenzoic acid, hydrogen peroxide (30 wt%), N-hydroxysuccinimide (NHS), methoxy poly(ethylene glycol) (mPEG), piperidine, succinic anhydride, trifluoroacetic acid (TFA), and triisopropylsilane (TPS) were obtained from Sigma-Aldrich without further purifications. The dialysis membrane with molecular weight cut-off (MWCO) 1 kDa and 3.5 kDa were acquired from Fisher Scientific. Reagents for cell culture, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium Eagle (MEM) and fetal calf serum were purchased from Life Technologies Holding Pte Ltd.

Characterization. Nuclear magnetic resonance (NMR) spectra were measured on Bruker

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AV 300 NMR system and chemical shifts (δ, ppm) were recorded by using internal reference tetramethyl silane (TMS). Mass spectra were performed with a Thermo LCQ Deca XP MAX (electrospray ionization measurement). Transmission electron microscopy (TEM) images were acquired at 100 kV with a FEI EM208S Transmission Electron Microscope (Philips). Powder X-ray diffraction pattern (XRD) was measured on a Philips X’Pert powder X-ray diffractometer. Fourier transformed infrared (FTIR) spectra were performed with a Shimadzu IRPrestige-21 FTIR spectrometer. UV-Vis absorption spectra were carried out on a Shimadzu UV/Vis/NIR spectrometer. Fluorescence spectra were recorded with a Shimadzu RF5301PC spectrometer. Platinum levels were quantified by an Agilent 7700 Inductively Coupled Plasma Mass Spectrometer (ICP-MS) or inductively coupled plasma optical emission spectrometer (ICP-OES). Dynamic light scattering (DLS) measurement was collected by a Malvern equipment. Flow cytometry was measured with a Fortessa X20 (3 laser) flow cytometer. The absorbance for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out using a Tecan's Infinite M200 microplate reader at wavelength of 490 nm.

Synthesis of CDs. Citric acid (2.10 g) and diethylenetriamine (3.50 g) were reacted in nitrogen atmosphere at 170 °C for 3 h. The mixture was cooled down to room temperature before adding a certain volume of water. The solution was dialyzed against water for 48 h by using a dialysis membrane with MWCO 3.5 kDa followed by freeze drying.

Synthesis of c,t,c-[PtCl2(OH)2(NH3)2]. cis-[PtCl2(NH3)2] (300.0 mg) was suspended in water (7.5 mL), and hydrogen peroxide (10.5 mL, 30% wt%) was added dropwise at 50 °C. After stirring for 2 h, the mixture was cooled down to room temperature. The solvent was reduced to about 2 mL by rotary evaporation, and the resulting solution was kept at 0 °C

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overnight. The pale yellow crystals were collected by filtration and washed with cold water and cold ether, which were then dried in vacuo. Yield: 53%.

Synthesis of c,c,t-[PtCl2(OH)(NH3)2(O2CCH2CH2CO2H] (Cisplatin(IV) Prodrug). c,t,c-[PtCl2(OH)2(NH3)2] (120.0 mg) and succinic anhydride (36.0 mg) were dissolved in dimethyl sulphoxide (DMSO, 2.0 mL), and the solution was stirred at room temperature for 24 h. The mixture was frozen dried and the solid was washed by cold acetone and diethyl ether to produce c,c,t-[PtCl2(OH)(NH3)2(O2CCH2CH2CO2H]. Yield: 62%.

1

H NMR:

(DMSO-d6): 5.91 (m, 6H, NH3), 2.39 (m, 4H, CH2). ESI-MS calcd: 434.13; found: 435.01 [M+H]+. FTIR spectra of cisplatin, c,t,c-[PtCl2(OH)2(NH3)2] and cisplatin(IV) prodrug are shown in the Supporting Information.

Synthesis

of

Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH.

The

peptide

Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH was prepared by employing Fmoc chemistry based solid phase peptide synthesis (SPPS) with 2-chlorotrityl chloride resin. The coupling reaction was performed with HBTU/DIPEA as coupling reagents at room temperature, and deprotection of Fmoc group was carried out in 20% (v/v) piperidine/N,N′-dimethylformamide (DMF) for 20 min. After the completion of coupling the last peptide Fmoc-Gly-OH, 2% TFA and

2.5%

TPS

in

dichloromethane

were

used

to

cleave

the

peptide

Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH from resin for several times. The cleavage mixture was collected and concentrated by rotary evaporation, followed by precipitation in cold ether. The peptide Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH was obtained by filtration, dried under vacuum and stored at -20 °C for further use. The molecular weight was determined by ESI-MS, calcd: 1305.45; found: 1305.44 (M+).

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Synthesis of mPEG-CHO. 4-Formylbenzoic acid (112.5 mg) was dissolved in DMF (0.5 mL) and dichloromethane (5 mL), followed by the addition of mPEG (300.0 mg), DCC (307.5 mg) and DMAP (45.0 mg). The mixture was stirred at room temperature for 24 h. Then, the solvent was removed using a rotary evaporator to afford white solid. The white solid was dissolved in dichloromethane and filtered through Buchner funnel to discard the precipitate dicyclohexylurea (DCU). Dichloromethane was removed via rotary evaporator and the product was then recrystallized using 2-propanol several times to give white solid of mPEG-CHO. mPEG-CHO was characterized by 1H NMR and FTIR (shown in the Supporting Information) and stored at room temperature for further use.

Synthesis of CDs-RGD(Pbf). Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH (3.3 mg), NHS (0.3 mg) and EDC•HCl (0.5 mg) were stirred in DMSO (0.5 mL) at room temperature for 30 min. Then, CDs (9.8 mg) in DMSO (1 mL) was added into the above solution. The mixture was stirred at room temperature for another 24 h and then dialyzed against water for 24 h by using a dialysis membrane with MWCO 3.5 kDa to remove the impurity. CDs-RGD(Pbf) was obtained after freeze drying and stored at -20 °C for further use.

Synthesis of CDs-RGD(Fmoc). The CDs-RGD(Pbf) obtained from the above synthesis was dissolved in solvent (1 mL) containing 95% TFA, 2.5% water and 2.5% TPS. The solution was left for stirring at room temperature for 2 h. Solvent was removed via rotary evaporator and the resultant was then dialyzed against water for 24 h by using a dialysis membrane with MWCO 1 kDa to remove the impurity. CDs-RGD(Fmoc) was obtained after freeze drying and stored at -20 °C for further use.

Synthesis of CDs-RGD(Fmoc)-Pt(IV). Cisplatin(IV) prodrug (4.3 mg), EDC•HCl (1.9

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mg) and NHS (1.1 mg) were dissolved in DMF (1 mL) before stirring at room temperature in dark condition for 12 h. The mixture was added into diethyl ether (40 mL), and the obtained solution was placed in a 4 oC refrigerator for 4 h. The solution was then centrifuged at 9000 rpm for 5 min to obtain the precipitate cisplatin(IV)-NHS. The precipitate was washed with diethyl ether for three times. The obtained CDs-RGD(Fmoc) and cisplatin(IV)-NHS were dissolved in DMSO (1 mL) containing DIPEA (3.4 µL). The mixture was stirred at room temperature in dark condition for another 24 h, and then dialyzed against water for 24 h by using a dialysis membrane with MWCO 1 kDa to remove the impurity. CDs-RGD(Fmoc)-Pt(IV) was obtained after freeze drying and stored at -20 °C for further use.

Synthesis of CDs-RGD-Pt(IV). The obtained CDs-RGD(Fmoc)-Pt(IV) was dissolved in solvent (1 mL) containing 80% DMF and 20% piperidine. The mixture was stirred at room temperature in dark condition for 20 min, and then dialyzed against water for 24 h by using a dialysis membrane with MWCO 1 kDa to remove the impurity. CDs-RGD-Pt(IV) was obtained after freeze drying and stored at -20 °C for further use.

Synthesis of CDs-RGD-Pt(IV)-PEG. The obtained CDs-RGD-Pt(IV) and mPEG-CHO (5.4 mg) were dissolved in DMSO (1 mL). The mixture was heated and maintained at 40 °C with stirring in dark condition for 24 h, and then dialyzed against water (pH = 8-9 adjusted by NaOH) by using a dialysis membrane with MWCO 3.5 kDa for 24 h to remove the impurity. CDs-RGD-Pt(IV)-PEG was obtained after freeze drying and stored at -20 °C for further use.

Quantum Yield Measurement. The quantum yields of CDs and CDs-RGD-Pt(IV)-PEG were determined by using quinine sulfate as a reference (54%, 0.1 M H2SO4 as solvent). The

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formula for calculating quantum yield (QY) is: QYx = QYref (Ix / Iref) (ηx2 / ηref2) (Aref / Ax) where x stands for the sample, ref stands for the standard compound quinine sulfate, I means the integrated fluorescence intensity, η indicates the refractive index for the solvent, and A means the absorbance at the excitation wavelength (less than 0.1 to decrease reabsorption effect).51

Confocal Laser Scanning Microscopy (CLSM). The human breast adenocarcinoma epithelial cell line MDA-MB-231 (ECACC) was cultured in DMEM with the addition of 10% fetal calf serum at 37 °C in a humidified incubator having 5% CO2. The human breast adenocarcinoma epithelial cell line MCF-7 (ECACC) was cultured in MEM with supplementation of 10% fetal calf serum at 37 °C in a humidified atmosphere having 5% CO2. MDA-MB-231 or MCF-7 cells at a density of 1×105 were cultured using a µ-dish with diameter of 35 mm and plastic-bottom (ibidi GmbH, Germany) at 37 °C in a humidified incubator with 5% CO2 for 24 h. Then, the medium was replaced by DMEM or MEM with pH 6.8 or pH 7.4 containing CDs-RGD-Pt(IV)-PEG before the maintenance at 37 °C in a humidified environment with 5% CO2 for another 2 h. After being rinsed by PBS twice, the MDA-MB-231 or MCF-7 cells were used for CLSM imaging, in which different measurement parameters were used under different excitation wavelengths (405, 488 and 543 nm) in order to show the CLSM images clearly. In addition, the same measurement parameters were utilized under the same excitation wavelengths for comparison.

Flow Cytometry Analysis. MDA-MB-231 or MCF-7 cells were cultured in 6-well plates with density of 5×105 per well at 37 °C in a humidified incubator with 5% CO2 for 24 h. Then,

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the medium was replaced by DMEM or MEM with pH 6.8 or pH 7.4 containing CDs-RGD-Pt(IV)-PEG at 37 °C in a humidified environment with 5% CO2 for designated time. After being washed and trypsinized, the MDA-MB-231 or MCF-7 cells were analyzed using flow cytometer in Indo 1 violet channel with an excitation wavelength of 355 nm and an emission wavelength range of 450/50 nm.

Intracellular Platinum Level Determination. MDA-MB-231 or MCF-7 cells were seeded in 12-well plates with density of 3×105 per well at 37 °C in a humidified incubator with 5% CO2 for 24 h. Then, the medium was replaced by DMEM or MEM with pH 6.8 or 7.4 containing CDs-RGD-Pt(IV)-PEG for 2 h, respectively. After the cells were washed, trypsinized and digested, intracellular platinum levels were determined by ICP-OES.

In Vitro Cytotoxicity. The MTT assay was used to evaluate the cytotoxicity of CDs-RGD-Pt(IV)-PEG. The MDA-MB-231 or MCF-7 cells were cultured in 96-well plates with density of 4×103 cells per well within DMEM or MEM (100 µL) at 37 °C in a humidified incubator with 5% CO2 for 24 h. Then, the medium was replaced by DMEM or MEM with pH 6.8 or pH 7.4 containing CDs-RGD-Pt(IV)-PEG at varied concentrations before the maintenance at 37 °C in a humidified environment with 5% CO2 for another 2 h. After being substituted with fresh DMEM or MEM, the MDA-MB-231 or MCF-7 cells were incubated at 37 °C for another 72 h. Then, the MDA-MB-231 or MCF-7 cells were rinsed using PBS, followed by being incubated with 20% MTT in DMEM or MEM at 37 °C for 5 h. After discarding the medium, DMSO (100 µL) was used to lyse the MDA-MB-231 or MCF-7 cells. The absorbance at 490 nm was recorded by Tecan's Infinite M200 microplate reader. Every experiment was performed thrice and the cell viability was calculated by referring to

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the control group without the sample treatment.

RESULTS AND DISCUSSION Preparation and Characterization. CDs were synthesized by a thermal pyrolysis method with citric acid as a carbon source and diethylenetriamine as a surface passivation agent, and purified through freeze drying after dialysis against water to remove small molecules.52 The morphology and structure of CDs were verified by several analyses. TEM characterization (Figure 1) shows that CDs have a uniform dispersion with particle diameters of 5-8 nm. The powder XRD patterns of the CDs (Figure S1) reveal three broad diffraction peaks centered at 0.40 nm, 0.30 nm and 0.22 nm, which are indicative of disordered carbon atoms and the (002) and (100) graphite lattice spacing, respectively. FTIR spectrum (Figure S2) was employed to demonstrate the surface functional groups on CDs. The broad vibration bands at 3000-3500 cm-1 correspond to O-H and N-H stretching vibration, and the vibration bands at 1569 cm-1 and 1354 cm-1 are from bending vibration of N-H and stretching vibration of C-N respectively, which identify the presence of many amino groups on the surface of CDs. The existence of surface amino groups on CDs was also proven by the formation of the Ruhemann's purple when CDs were reacted with ninhydrin, and the quantity of amino groups was 1.27 µmol/mg by measuring the absorbance of Ruhemann's purple at 570 nm with alanine as the reference (Figure S3). In the UV-Vis spectrum (Figure 2a), CDs show two absorption bands at 240 nm and 343 nm, which are originated from the π-π* transition of the aromatic ring structure and the n-π* transition of the C=O group, respectively. In the fluorescence spectra (Figure 2b), CDs present optimal emission wavelength at 458 nm under excitation

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wavelength of 360 nm with a quantum yield of 25.5%. The emission peak of CDs redshifts from 458 nm to 537 nm when increasing the excitation wavelength from 360 nm to 480 nm. This excitation-dependent fluorescence behavior is contributed to different band gaps from various surface states of CDs.53,54

Figure 1. TEM image of CDs.

Figure 2. (a) UV-Vis absorption spectrum and (b) excitation-dependent fluorescence spectra of CDs. In order to offer the CDs with therapeutic effects, a cisplatin(IV) prodrug with an axial carboxyl group was selected as the model drug, which was synthesized according to a

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Page 14 of 29

reported method (Figure S4).49,50 The cisplatin(IV) prodrug has low toxic side effects and can be activated to cytotoxic cisplatin specifically in reductive cytosol of cancer cells. To endow the

CDs

with

active

targeting

property,

the

peptide

sequence

Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH (Figure S5) with protection groups was synthesized manually using Fmoc-based solid-phase synthesis method. The molecular mass of this peptide was calculated to be 1305.45 and the measured value was 1305.44 by ESI-MS, confirming successful synthesis. By processing Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH with TFA/H2O/TPS, the protection groups except Fmoc group were deprotected to produce Fmoc-GRGDSGGGG-OH (Figure S6). The RGD peptide with NH2-GRGDSGGGG-OH sequence could finally be obtained through the deprotection of Fmoc group using DMF/piperidine, and its structure was also verified by ESI-MS (Figure S7). The mPEG-CHO polymer (Figure S8) with benzaldehyde group was synthesized for protecting the RGD peptide to maintain stealth property in physiological condition, and its structure was confirmed by 1H NMR spectrum (Figure S8). The designed drug nanocarrier CDs-RGD-Pt(IV)-PEG was prepared by first conjugating the peptide Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH with CDs through a typical EDC/NHS

amidation

reaction

between

the

carboxylic

acid

group

of

Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH and a portion of amino groups on CDs. After deprotecting the protection groups except Fmoc group, the cisplatin(IV) prodrug was loaded using the remaining amino groups on CDs. Following the deprotection of Fmoc group to expose the amino groups, mPEG-CHO was introduced to protect the RGD peptide by forming a benzoic-imine bond to produce CDs-RGD-Pt(IV)-PEG. As shown in Figure 3,

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CDs-RGD-Pt(IV)-PEG displays two special peaks of amide bands I and II at 1654 and 1558 cm-1 respectively, proving the presence of RGD peptide on the surface. The strong absorption peak at 1121 cm-1 is originated from the C-O stretching vibration of PEG, confirming the functionalization of the mPEG-CHO polymer. The platinum loading percentage of CDs-RGD-Pt(IV)-PEG was determined to be 4.1 wt% by ICP-MS. DLS data (Figure S9) of CDs-RGD-Pt(IV)-PEG present relatively larger diameter of 31 nm as compared with the size of CDs, further supporting the successful preparation of the drug nanocarrier. The UV-Vis absorption spectrum of CDs-RGD-Pt(IV)-PEG (Figure S10) shows two absorption bands at 250 nm and 351 nm for the π-π* transition of the aromatic ring structure and the n-π* transition of the C=O group, respectively. The fluorescence spectra of CDs-RGD-Pt(IV)-PEG (Figure S11) are also excitation-dependent, with redshifted emission peak from 461 nm to 534 nm under increasing excitation wavelength from 360 nm to 480 nm. As compared with the spectral properties of CDs, the absorption bands and the emission peaks of CDs-RGD-Pt(IV)-PEG show some shifts, which are contributed to the varied surface states after the functionalization of RGD peptide, cisplatin(IV) prodrug and mPEG-CHO on CDs.

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Figure 3. FTIR spectra of CDs, RGD (NH2-GRGDSGGGG-OH), cisplatin(IV) prodrug, mPEG-CHO, and CDs-RGD-Pt(IV)-PEG.

Cellular Uptake Measured by CLSM and Flow Cytometry. To explore the tumor extracellular

acidic

condition

triggered

targeting

ability

of

the

nanocarrier,

CDs-RGD-Pt(IV)-PEG was incubated with MDA-MB-231 and MCF-7 breast cancer cells, followed

by

CLSM

imaging

for

the

multicolor fluorescence

characteristics

of

CDs-RGD-Pt(IV)-PEG under different excitation wavelengths of 405, 488 and 543 nm (Figure S12). Owning to the targeting interaction between RGD containing peptide and integrin αvβ3, MDA-MB-231 cells were used as integrin-positive cancer cells for the overexpression of integrin αvβ3 on the cellular membrane, while MCF-7 cells were chosen as the negative control for low expressed integrin αvβ3. As shown in CLSM imaging results (Figure 4a), MDA-MB-231 cells showed faint blue, green and red fluorescence under excitation wavelength of 405 nm, 488 nm and 543 nm in physiological condition (pH 7.4), respectively. This observation indicates that only few CDs-RGD-Pt(IV)-PEG was endocytosed by MDA-MB-231 cells, because the benzoic-imine bond was not hydrolyzed at pH 7.4 and RGD peptide was still shielded by the PEG polymer. While at tumor extracellular environment (pH 6.8), MDA-MB-231 cells exhibited stronger blue, green and red fluorescence under different excitation wavelengths (Figure 4b). The enhanced endocytosis of the nanocarrier was achieved by the interaction between the RGD peptide and integrin αvβ3 due to the exposure of RGD peptide after the hydrolysis of the benzoic-imine bond at pH 6.8. In contrast, MCF-7 cells displayed weak blue, green and red fluorescence both at pH 7.4 and

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6.8 (Figure 4c and 4d). The low uptake of the nanocarrier at pH 7.4 by MCF-7 cells is resulted from the shielding of the RGD peptide by the PEG polymer, whereas at pH 6.8, the exposed RGD peptide cannot function effectively due to the low level of integrin αvβ3 expression on the cellular membrane of MCF-7 cells.

Figure 4. CLSM images of (a,b) MDA-MB-231 and (c,d) MCF-7 cells upon the treatment with CDs-RGD-Pt(IV)-PEG at pH (a,c) 7.4 and (b,d) 6.8 at 37 °C under excitation wavelength of 405 nm, 488 nm and 543 nm.

Flow cytometry analysis was also performed to quantitatively investigate the cellular 17



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uptake behavior of CDs-RGD-Pt(IV)-PEG by MDA-MB-231 and MCF-7 cancer cells. The fluorescence of CDs-RGD-Pt(IV)-PEG was recorded in Indo 1 violet channel with an excitation wavelength of 355 nm and an emission wavelength range of 450/50 nm. No obvious difference in the fluorescence intensity of MDA-MB-231 or MCF-7 cells was observed between pH 7.4 and pH 6.8 (Figures S13 and S14). When MDA-MB-231 cells were incubated with CDs-RGD-Pt(IV)-PEG, the cellular uptake was apparently higher at pH 6.8 than that at pH 7.4 (Figure 5a). The mean fluorescence intensity of CDs-RGD-Pt(IV)-PEG in MDA-MB-231 cells at pH 6.8 was 1.9-fold higher than that at pH 7.4 (Figure S15). While for MCF-7 cells, less uptake of CDs-RGD-Pt(IV)-PEG was observed (Figure 5b) and the mean fluorescence intensity has no significant difference between pH 7.4 and pH 6.8 (Figure S16). In addition, an increased intracellular platinum level was detected for MDA-MB-231 cells treated with CDs-RGD-Pt(IV)-PEG at pH 6.8 (Figure S17). The flow cytometry and intracellular platinum level results were in consistent with the CLSM imaging observations, demonstrating that CDs-RGD-Pt(IV)-PEG could be internalized effectively by integrin αvβ3 overexpressed MDA-MB-231 cancer cells with tumor extracellular microenvironment of pH 6.8.

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Figure 5. Flow cytometry results of (a) MDA-MB-231 and (b) MCF-7 cells upon the incubation with CDs-RGD-Pt(IV)-PEG at pH 7.4 and 6.8. Control samples correspond to MDA-MB-231 and MCF-7 cells without any treatment. In Vitro Cytotoxicity Study. The anticancer efficiency of CDs-RGD-Pt(IV)-PEG was evaluated by MTT assay using MDA-MB-231 and MCF-7 cancer cells. The CDs alone showed negligible toxicity towards MDA-MB-231 and MCF-7 cells even at a high nanoparticle concentration of 200 µg/mL (Figures S18 and S19). When MDA-MB-231 cells were incubated with CDs-RGD-Pt(IV)-PEG at pH 6.8, much higher cytotoxicity was observed than that at pH 7.4 under the same nanocarrier concentration (Figure 6a). On the contrary, there was no obvious difference in the cytotoxicity for MCF-7 cells incubating with CDs-RGD-Pt(IV)-PEG at pH 7.4 and pH 6.8 (Figure 6b). The enhanced therapeutic efficiency of CDs-RGD-Pt(IV)-PEG to integrin αvβ3 overexpressed MDA-MB-231 cancer cells at tumor extracellular condition was contributed to its increased uptake through receptor-mediated endocytosis with the aids of the deprotection of PEG polymer and the exposure of RGD targeting ligand at pH 6.8. 19



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Figure 6. Cytotoxicity assay of CDs-RGD-Pt(IV)-PEG under different concentrations on (a) MDA-MB-231 and (b) MCF-7 cells at pH 7.4 and 6.8 at 37 °C.

CONCLUSIONS In summary, multicolor imaging-guided and pH/redox dual-responsive drug nanocarrier CDs-RGD-Pt(IV)-PEG

has

been

successfully

developed

for

tumor

extracellular

microenvironment triggered targeting towards integrin αvβ3 overexpressed cancer cells. The PEG

protection

of

RGD

targeting

ligand

via

a

benzoic-imine

bond

enables

CDs-RGD-Pt(IV)-PEG to present increased internalization and enhanced therapeutic efficiency at tumor extracellular pH, as well as lowered uptake and less cytotoxicity in physiological condition for integrin αvβ3 overexpressed cancer cells. On the other hand, the nanocarrier shows less cytotoxicity to cells with low integrin αvβ3 expression level. Thus, the CDs-RGD-Pt(IV)-PEG drug nanocarrier offers a new opportunity for developing safe and efficient anticancer therapeutics. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at 20


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http://pubs.acs.org. Powder XRD pattern and FTIR spectrum of CDs; linear curve for amino group quantitative determination; FTIR spectra of cisplatin(IV) prodrug; chemical structures and ESI-MS

spectra

of

Fmoc-GR(Pbf)GD(OtBu)S(tBu)GGGG-OH,

Fmoc-GRGDSGGGG-OH and NH2-GRGDSGGGG-OH; synthetic route and 1H NMR spectrum of mPEG-CHO; DLS result, UV-Vis absorption and fluorescence spectra of CDs-RGD-Pt(IV)-PEG; flow cytometry results and intracellular platinum levels of MDA-MB-231 and MCF-7 cells at different pH or treated with CDs-RGD-Pt(IV)-PEG; MTT assay for CDs against MDA-MB-231 or MCF-7 cells (PDF). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This research is supported by the NTU-A*Star Silicon Technologies Centre of Excellence under program grant no. 11235100003 and the NTU-Northwestern Institute for Nanomedicine. REFERENCES (1) Dawson, L. A.; Jaffray, D. A. Advances in Image-Guided Radiation Therapy. J. Clin. Oncol. 2007, 25, 938-946. (2) Melancon, M. P.; Zhou, M.; Li, C. Cancer Theranostics with Near-Infrared

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TOC FIGURE

29


Dual-Responsive Carbon Dots for Tumor Extracellular

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