Research Article www.acsami.org
Porous Covalent Triazine Polymer as a Potential Nanocargo for Cancer Therapy and Imaging Arunkumar Rengaraj,†,⊥ Pillaiyar Puthiaraj,‡,⊥ Yuvaraj Haldorai,§ Nam Su Heo,† Seung-Kyu Hwang,† Young-Kyu Han,§ Soonjo Kwon,∥ Wha-Seung Ahn,*,‡ and Yun Suk Huh*,† †
Department of Biological Engineering, Biohybrid Systems Research Center (BSRC), Inha University, Incheon, 402-751, Republic of Korea ‡ Department of Chemistry and Chemical Engineering, Inha University, Incheon 402-751, Republic of Korea § Department of Energy and Materials Engineering, Dongguk UniversitySeoul, Seoul, 100-715, Republic of Korea ∥ Department of Biological Engineering, Integrated Tissue Culture Laboratory, Inha University, Incheon, 402-751, Republic of Korea S Supporting Information *
ABSTRACT: A microporous covalent triazine polymer (CTP) network with a high surface area was synthesized via the Friedel−Crafts reaction and employed as a potential transport system for drug delivery and controlled release. The CTP was transformed to the nanoscale region by intense ultrasonication followed by filtration to yield nanoscale CTP (NCTP). This product showed excellent dispersibility in physiological solution while maintaining its chemical structure and porosity. An anticancer drug, doxorubicin (DOX), was loaded onto the NCTP through hydrophobic and π−π interactions, and its release was controlled at pH 4.8 and 7.4. The NCTP showed no toxicity toward cancer or normal cells, but the NCTP-DOX complex showed high efficacy against both types of cells in vitro. In-vitro cell imaging revealed that NCTP is a potential material for bioimaging. The potency of NCTP on cellular senescence was confirmed by the expression of senescence associated marker proteins p53 and p21. These results suggest that NCTP can be used as a new platform for drug delivery and imaging with potential applications in diagnosis and therapy. KEYWORDS: covalent triazine polymer, porous material, fluorescence, biocompatible, drug delivery
■
INTRODUCTION Cancer has a major impact on society, with more than 15 million individuals expected to be afflicted by 2020. The world health organization stated that there are over 10 million new cases and 6 million deaths caused by cancer annually.1 For the past few years, there has been significant progress in the treatment of cancer; however, adequate therapy remains elusive owing to the inadequate strategies for addressing aggressive metastasis, late diagnosis, and a lack of clinical procedures for overcoming multidrug resistant cancer.2 The integration of nanotechnology and medicine allow us to control the fundamental molecular structure of cancer cells. Nanocarriers have been tailored to interact with tumors and inflammation sites that have permeable vasculature. Recently, a wide range of porous materials including activated carbons, zeolite, metal−organic frameworks (MOFs), porous organic frameworks (POFs), and amine modified porous silica have been investigated for their potential application in drug delivery.3−7 Some of these materials have been utilized in the encapsulation of efficient drugs to avoid the “burst effect” and to enable bioimaging.8−10 Horcajada et al.11 demonstrated the drug delivery application of various iron-MOFs, while Zhuang et al.12 reported the potential application of zinc-MOF as a drug © 2016 American Chemical Society
carrier. However, these MOFs are unstable in water because of the decomposition of coordination bonds and frameworks made by biologically toxic metals.13,14 To overcome these problems, researchers have recently focused on highly stable POFs. POFs are a new class of emerging lightweight materials in which the constituent blocks are formed via strong covalent bonds. POFs have received increasing interest because they have potential widespread applications in drug delivery owing to their high specific surface area, porous structures, easy synthesis, low cost, high nitrogen contents, and high thermal and chemical stability.15 CTP is a subclass of POFs that has high nitrogen contents with free lone pairs of electrons, which provides high electron density in the network to interact with drug compounds. Zhao et al.16 reported the possibility of using two-dimensional POF as a carrier material for anti-inflammatory drug delivery. Doxorubicin (DOX), which is broadly used as a drug molecule in cancer therapy, functions via inhibition of Received: January 8, 2016 Accepted: March 21, 2016 Published: March 21, 2016 8947
DOI: 10.1021/acsami.6b00284 ACS Appl. Mater. Interfaces 2016, 8, 8947−8955
Research Article
ACS Applied Materials & Interfaces bimolecular synthesis by inserting DOX into DNA.17 When compared to other cancer drugs, DOX is highly efficient at inducing apoptosis and senescence.18 However, it causes severe side-effects because it kills both cancerous cells and healthy cells. Accordingly, nanotechnology has attracted a great deal of interest because of its potential for development of novel drug delivery systems that target cancerous cells directly while causing minimal damage to normal cells and avoiding burst release of drugs into the circulatory system. Cellular senescence can be considered a powerful mechanism to suppress tumors and withdraw cells with irreparable DNA damage from the cell cycle.19,20 These anti progress programs are prompted by different tumor suppressor genes in response to potential oncogenic signals. The p53 and p21 proteins are important mediators of quiescence, apoptosis and senescence, and this senescence can be reversed upon subsequent inactivation of p53. Senescent cells can be confirmed by their physiological characteristics (increased cell size and flat vacuolated morphology), or by senescence-associated β-galactosidase assay (SA-β-gal).21 In this study, CTP was successfully synthesized via the Friedel−Crafts reaction and used as a carrier for controlled drug release. The nanoscale CTP (NCTP) exhibited good dispersibility in physiological solution. The toxicity of the NCTP was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which clearly showed the nontoxic nature of the material. DOX, an aromatic waterinsoluble cancer drug, was loaded onto the NCTP by physisorption, and its ability to deliver DOX was examined. In addition, drug transfer efficacy in cancer cells (HeLa) and normal cells (COS-7) was studied by confocal imaging and biotransmission electron microscopy (bio-TEM).
■
NCTP, the unbound DOX in the supernatant was determined by UV−visible spectroscopy (UV−vis) based on comparison to a calibration curve of DOX standard solution. For the drug release process, two identical NCTP-DOX samples (2 mg) were immersed in a 20 mL PBS (pH = 7.4 and 4.8) solution and shaken while being incubated at 37 °C for 24 h. After incubation, 4 mL of PBS was removed at different time intervals. The samples were then centrifuged and the same amount of corresponding buffer was added. The DOX release rate was determined by the absorbance of DOX (λab = 480 nm). All the measurements were repeated three times. Estimation of Cellular Killing Ability. Different concentrations of DOX and NCTP-DOX (0, 2, 5, 10, 15, and 20 μg mL−1) diluted with DMEM medium were added to 9-well plates containing HeLa and COS-7 cells. The cells were then incubated for 24 h at 37 °C, after which the absorbance at 480 nm was measured using a VersaMax ELISA microplate reader. The relative cell viability (%) was estimated from Atest/Acontrol × 100%, where Atest and Acontrol are the absorbance of the wells with NCTP and control (without NCTP), respectively. Bio-TEM Analysis. Bio-TEM analysis was used to determine the intracellular distribution of DOX and NCTP−DOX. Samples were prepared as follows: after 2 h of incubation, cells were washed with PBS to remove the NCTP from medium. The cells were then washed with 0.1 M cacodylate buffer for 1 h, after which they were fixed in a solution containing 2.5% glutaraldehyde overnight. After postfixation, the samples were processed by adding 1% osmium tetroxide for 2 h. Following dehydration with alcohol, the samples were fixed in an epoxy resin, dried at 60 °C for 24 h, and then cut into thin sections using a microtome (Reichert no. 318423, Austria). Finally, the samples were stained using 2% uranyl acetate and 1% lead citrate. Confocal Fluorescence Imaging. COS-7 and HeLa cells were incubated with NCTP, DOX, and NCTP-DOX (containing 100 μg mL−1 DOX) at 37 °C for 24 h. After being washed with PBS three times the cells were examined using a confocal fluorescence microscope (Nikon C2). Senescence Chromogenic Assay. The assay was prepared according to the manufacturer’s instructions (Sigma-AldrichCS0030). Subconfluent HeLa cells in 6 mm plates were washed with PBS for 60 s, and then fixed using a fixing solution for 5 min. Following fixation, the plates were washed with PBS to remove the unbounded fixing solution, after which 2 mL of staining solution was added to the cells, and they were incubated overnight. Next, the plates were washed with a washing kit containing a small amount of methanol and PBS, after which the cells were dried at room temperature and observed under a light microscope. The expression of proteins was evaluated by Western blotting, after which they were transferred to nitrocellulose membranes that were then stained with consecutive monoclonal antibodies of the specific protein. Finally, the membranes were developed in X-ray film under UV irradiation for further study.23 Characterization. Powder X-ray diffraction (XRD) was measured using a Rigaku diffractometer (Cu Kα radiation), while Fourier transform infrared (FTIR) spectroscopy was recorded on a VERTEX 80 V spectrometer (Bruker) under ambient conditions. Elemental analysis was conducted using a Flash 1112 Thermo scientific elemental analyzer. The morphology of the polymer sample was observed by field emission scanning electron microscopy (FESEM, HITACHI S4300). TEM analysis was conducted at an acceleration voltage of 200 kV (JEOL, JEM-2010F). Thermogravimetric analysis (TGA, TG 209 F3 tarsus) was conducted at a heating rate of 5 °C/min under an argon atmosphere. The nitrogen sorption isotherm was performed at 77 K on a BELsorp-Max apparatus. UV−vis analysis was measured on a Jasco V-650 spectrophotometer. Room temperature fluorescence was measured on a Jasco FP−6500 spectrofluorometer. Bio-TEM analysis was conducted using a Philips CM-10 electron microscope. Zeta potential and dynamic light scattering measurements were conducted on a Malvern Zetasizer Nano instrument (laser source of 633 nm at room temperature).
MATERIALS AND METHODS
Materials. Cyanuric chloride, biphenyl, dichloromethane (DCM), anhydrous aluminum chloride, phosphate buffered saline (PBS), DOX, MTT assay (98%), Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and all other reagents mentioned in this article were purchased from Sigma−Aldrich and used without purification. Synthesis of Covalent Triazine Polymer. CTP was prepared as previously described.22 Cyanuric chloride (1.48 g, 8 mmol) and biphenyl (1.85 g, 12 mmol) were added to a round-bottom flask containing DCM (100 mL) at room temperature. Anhydrous aluminum chloride (3.00 g, 24 mmol) was added slowly to the above mixture at room temperature as a catalyst, and the reaction was then carried out at 70 °C. After 16 h, the resulting product was collected by filtration and subsequently washed with DCM, methanol, and water several times to completely remove the unreacted starting materials and catalyst. Finally, the collected CTP was dried in a vacuum oven at 120 °C for 12 h. Cytotoxicity of NCTP. In-vitro cytotoxicity was evaluated by an MTT assay of a fibroblast-like cell line derived from monkey kidney tissue (COS-7 cells) and the human cervix adenocarcinoma cell line (HeLa cells). The cells were distributed into 9-well plates at a density of 1 × 104 cells per well and incubated at 37 °C for 24 h under 95% relative humidity (with 5% CO2), after which each well was washed with 0.01 M phosphate buffer solution (PBS, pH = 7.4). Various concentrations of NCTP (5, 50, 100, 250, and 500 μg) were then added with DMEM to the wells. After 24 h, an MTT assay was conducted. Drug Loading and Release Behaviors. In a typical experiment, 400 μg of NCTP was soaked in a 4 mL PBS with various concentrations (50−500 μM) of DOX for 24 h at room temperature. The DOX loaded NCTP was then centrifuged, washed with PBS, and freeze-dried for 24 h. To estimate the amount of DOX loading into 8948
DOI: 10.1021/acsami.6b00284 ACS Appl. Mater. Interfaces 2016, 8, 8947−8955
Research Article
ACS Applied Materials & Interfaces Scheme 1. Synthesis of NCTP and Encapsulation of DOX
Figure 1. (a) FT-IR, (b) N2 sorption isotherm, (c) zeta potential, and (d) UV−visible and fluorescence spectra of NCTP.
■
RESULTS AND DISCUSSION CTP was prepared via the Friedel−Crafts reaction using cyanuric chloride and biphenyl as the starting materials (Scheme S1 of the Supporting Information, SI). A total of 200 mg CTP was dispersed in water under sonication for 30 min (ultrasonic generator (Branson S-450A). After 30 min, the solution was passed through a 0.2 μm polyvinylidene fluoride filter to separate the NCTP. The amount of NCTP collected after filtration was found to be ∼50 mg (25% of yield). The
schematic representation for the synthesis of NCTP and encapsulation of the drug is presented in Scheme 1. The FT-IR spectrum of NCTP is shown in Figure 1a. The bands at 1610, 1519, and 1369 cm−1 were assigned to CC, CN, and CN stretching vibrations of the triazine rings in the polymer network, respectively. The disappearance of CCl stretching vibration (850 cm−1) indicated the complete substitution of cyanuric chloride with biphenyl, signifying the successful preparation of polymer. Figure S1a illustrates the 8949
DOI: 10.1021/acsami.6b00284 ACS Appl. Mater. Interfaces 2016, 8, 8947−8955
Research Article
ACS Applied Materials & Interfaces
Figure 2. SEM and TEM images of CTP (a, d) before sonication, (b, e) after sonication, and (c, f) after filtration.
Figure 3. Cytotoxicity study of the NCTP. Cell viability of (a) COS-7 and (b) HeLa cells.
NCTP was calculated to be 78% based on the ratio of the micropore volume to the total pore volume (V0.1/Vtot), indicating that most of the pores were microporous in nature. The NCTP showed a pore size distribution of 1.21 nm, which was consistent with a previous investigation of the same analogues.24 The stability of NCTP in the colloidal system was investigated using zeta potential as a function of pH and time, and the observed results were shown in Figure 1c and Table S2. The zeta potential of NCTP was about −20 mV at pH 2, which increased to −19 mV at pH 5, then decreased constantly with increasing pH, reaching −35 mV at pH 11. The increasing zeta potential may be attributed to the protonation of NCTP, whereas the decreasing zeta potential with increasing pH over 5 was due to deprotonation of the network. These results clearly showed that the NCTP formed a stable colloidal system at pH 2−11. Conversely, the as-synthesized CTP showed a zeta potential value of close to zero, which resulted in an unstable colloidal system (Figure S2). Figure 1d showed the absorption, excitation, and emission spectra of the NCTP. As shown in Figure 1d, the NCTP exhibited a strong emission band at 430 nm when it was excited at 370 nm. As the excitation wavelength changed to 380 nm, the corresponding emission was shifted (red shift) to 445 nm (Figure 1d inset).
PXRD patterns of CTP. The broad diffraction peaks observed at 2θ values of 11.4°, 21.7°, and 43.3° indicated the amorphous nature. Examination of thermal stability of the CTP by TGA from 25−700 °C under an argon atmosphere is shown in Figure S1b. The first weight loss of about 10% was observed at below 120 °C in the TGA curve due to the loss of water. The second weight loss occurred at 350 °C−600 °C, which was attributed to the decomposition of CTP. The residual mass reached 0% at around 620 °C, indicating the absence of aluminum. Elemental analysis of CTP showed that the C, N, and H contents were 80.86, 12.15, and 3.74%, respectively, which were in close agreement with the theoretical values (C = 82.33, N = 13.72, and H = 3.95%). Following the conversion of CTP into NCTP there was no change in the elemental composition of the material (Table S1). The porous properties of the NCTP were determined from the N2 sorption isotherm at 77 K up to 1 bar (Figure 1b). The sample was activated at 130 °C under vacuum for 10 h prior to measurement. According to the IUPAC classification, NCTP displayed a type I isotherm and exhibited a steep uptake in the low pressure region (0−0.1 bar), indicating a microporous nature of the material. The BET surface area of NCTP was 1220 m2/g, which was evaluated from the 0.01 < P/P0 < 0.05 region of the adsorption curve. The level of microporosity of 8950
DOI: 10.1021/acsami.6b00284 ACS Appl. Mater. Interfaces 2016, 8, 8947−8955
Research Article
ACS Applied Materials & Interfaces
Figure 4. Adsorption, release, and mechanism of DOX from NCTP-DOX complex. (a) DOX loading efficiency on NCTP, (b) drug release rate at pH 4.8 and 7.4, and (c) mechanism of DOX release from NCTP-DOX.
adsorbed initially increased to 25 μg, after which it became saturated. After encapsulation of DOX onto the NCTP, the morphology and particle size was investigated by TEM and DLS analysis. The TEM image (Figure S4a) indicated that the particle size of NCTP-DOX was increased slightly (60−80 nm) relative to the NCTP. DLS analysis (Figure S4b) showed a distribution of NCTP-DOX particles with a size of 65 nm. We also analyzed the fluorescence properties of DOX and NCTPDOX (Figure S5). When the DOX was excited at 480 nm, strong emission peaks were observed at 558, 592, and 640 nm, whereas the emission peaks of NCTP-DOX were quenched upon encapsulation of DOX onto the NCTP due to hydrophobic and π−π interactions. The stability of the NCTP-DOX complex at different times and pH were investigated by zeta potential analysis and the results are shown in Table S2. The DOX release rate from NCTP was evaluated by incubating 100 μg/mL of the NCTP-DOX at pH 4.8 and 7.4 at 37 °C, after which the percentage of DOX remaining in the NCTP was determined by UV−vis spectroscopy. The DOX release profile showed a rapid release from NCTP in the first 24 h, after which it was released slowly over a period of time (Figure 4b). About 60% of the DOX was released from NCTP at pH 7.4, whereas approximately 80% was released at pH 4.8 over 48 h. This may be ascribed to the higher solubility and hydrophilic nature of DOX at pH 4.8 due to protonation, which minimized the hydrophobic interaction between DOX and NCTP.26 The stability of DOX in the NCTP-DOX complex at pH 7.4 was higher because of the strong noncovalent interaction within DOX and NCTP. Hence, a greater amount of DOX was released from NCTP at pH 4.8, which is similar to
The emission property of NCTP may be attributed to the extended π conjugation present in the structure, which is concordant with observations described in previous reports.25 The fluorescence property of NCTP ensures that no additional dye was required for tracking in the biological system. Figure 2 shows the SEM and TEM images of the CTP before (Figure 2a, d) and after sonication (Figure 2b, e). Following sonication, it was clear that the CTP was broken into different sizes. To obtain a uniform size, the dispersion was passed through a 0.2 μm PVDF filter (Figure 2c, f). The TEM image showed that the NCTP particles were relatively spherical, with a mean diameter of 50−70 nm. We also conducted DLS analysis to measure particle size distribution (Figure S3), which showed bimodal polymer particles with the smallest one having an average size of 50 nm, and a largest one of around 115 nm originating from particle aggregation. The particle size increased from 50 to 73 nm as time increased from 0 to 50 h. To evaluate the cytotoxicity of NCTP, an MTT cell viability assay was conducted using COS-7 and HeLa cells (both are transformed cell lines) with different concentrations of NCTP for 24 h (Figure 3). No significant loss of cell viability was observed, even at an NCTP concentration of 500 μg/mL. The cell viability of COS-7 and HeLa cells was decreased to 82 and 94%, respectively. We also evaluated the loading of DOX onto NCTP and the release behavior of DOX from the NCTP by UV−vis spectroscopy. Loading was achieved by stirring DOX with NCTP in PBS at 500 rpm, followed by centrifugation at 1000 rpm, washing with PBS and freeze-drying. We increased the DOX concentration in the range of 20 to 100 μg/mL in the presence of 100 μg/mL of NCTP. The amount of DOX adsorbed on the NTCP was shown in Figure 4a. The amount 8951
DOI: 10.1021/acsami.6b00284 ACS Appl. Mater. Interfaces 2016, 8, 8947−8955
Research Article
ACS Applied Materials & Interfaces
Figure 5. Changes in images of HeLa cells incubated with NCTP, NCTP-DOX, and DOX for 24 h. (a) Optical, (b) confocal, and (c) overlay.
Figure 6. Relative cell viability of (a) COS-7 and (b) HeLa cells treated with DOX and NCTP-DOX at various concentrations; Bio-TEM images of (c) HeLa cells, (d) NCTP-DOX, and (e) DOX treated HeLa cells after 6 h.
To investigate the cellular interactions of NCTP-DOX over HeLa and COS-7, the cells were incubated at 37 °C for 24 h and pH 7.4 with 100 μg/mL of NCTP and NCTP-DOX (80 μg/mL of NCTP, 20 μg/mL of DOX). Figure 5 shows the optical, fluorescence, and overlay images of HeLa cells incubated with NCTP, DOX, and NCTP-DOX for 24 h. Under 375 nm laser excitation, an intense red fluorescence signal was observed in the intracellular space of HeLa cells incubated with DOX and NCTP-DOX. In the case of NCTPDOX, the fluorescent signal was mostly localized inside the nuclei of the cells because of the accumulation of more DOX.
the microenvironments of intracellular lysosomes and endosomes or cancerous tissues. In addition, the DOX released from NCTP in pH 4.8 at 37 °C was investigated by fluorescence spectroscopy. As shown in Figure S6, the intensities of DOX peaks (558, 592, and 640 nm) increased gradually upon the release of DOX from NCTP up to 36 h; after which the fluorescence intensity remained constant. This affords a built-in mechanism that enables the effective release of drugs from NCTP.27 The overall drug delivery mechanism is presented in Figure 4c. 8952
DOI: 10.1021/acsami.6b00284 ACS Appl. Mater. Interfaces 2016, 8, 8947−8955
Research Article
ACS Applied Materials & Interfaces
Figure 7. Senescence β-galactosidase assay for HeLa cells treated with DOX and NCTP-DOX. Panels (a) to (c) represent micrographs before staining. Panels (d) to (f) represent micrographs staining with β-galactose. After incubating with β-galactose, the cells became senescence positive (blue color).
Figure 8. Senescence-related protein expression for HeLa cells treated with (a) DOX and (b) NCTP-DOX. mTOR (mammalian target of rapamycin), p53 and p21 (senescence related proteins), pRb (retinoblastoma protein), and β-actin (control).
incubation with DOX (Figure 6e). Since the intracellular compartments of cancer cells were acidic, NCTP released more DOX, which was transported to the nucleus via proteasomes. This ensured an adequate DOX concentration in the nucleus to induce more cancer cytotoxicity and prolonged senescence activity.28 The senescence of DOX and NCTP-DOX in HeLa cells was observed during 3 days of exposure. Both cells exhibited changes in their size and shape. After incubation with βgalactose, the cells became senescence positive (blue color). Figure 7 shows the β-galactosidase assay for nontreated HeLa cells (control), DOX, and NCTP-DOX-treated HeLa cells before and after staining. The results of NCTP-DOX-treated HeLa cells confirmed the presence of SA-β-gal enzyme, which converts β-galactose into its monosaccharides.29 The induction of cellular senescence and efficacy of NCTP can be confirmed by the expression of senescence associated marker proteins p53 and p21. Figure 8 shows that both DOX and NCTP-DOX induced senescence (p53 and p21 were positive) in HeLa cells, but the expression of several proteins differed. Phospho-mTOR (pmTOR) is responsible for maintaining the cell cycle. In cancer cells, the expression of p-mTOR was significantly higher than that of the normal cells. In this study, NCTP-DOX greatly reduced (or inhibited) the expression of p-mTOR compared to
The optical and confocal microscopic images of COS-7 cells incubated with NCTP, DOX, and NCTP-DOX for 24 h are shown in Figure S7. The cytotoxic effects of DOX and NCTP-DOX at different concentrations on COS-7, HeLa cells after 24 h are shown in Figure 6a, b. The NCTP-DOX showed a higher cytotoxicity than DOX throughout the concentration range (2−20 μg/mL). When cells were cultured with DOX at a concentration of 20 μg mL−1, the cell viabilities were around 30%, while culture with NCTP-DOX resulted in cell viabilities of 5 and 10% for the COS-7 and HeLa cells, respectively. These results indicated that the NCTP-DOX can deliver more DOX into the cells. The cell order and distribution of NCTP in HeLa cells were determined by Bio-TEM analysis (Figure 6c−e). Figure 6c shows the image of the HeLa cells without NCTP-DOX (control sample). The image of HeLa cells incubated with NCTP-DOX for 6 h shows the distribution of NCTP into the cells, as well as the effects of DOX on the cancer cells. The results clearly revealed a well-ordered cell structure including the condensed form of the nucleus and a very small amount of peroxisomes in the cytoplasm. The NCTP-DOX was dispersed throughout the cytoplasm, the nuclear membrane had started to degrade, chromosomal material began fragmenting, and many peroxisomes were formed in the cell (Figure 6d). However, the HeLa cells were completely destroyed after 8953
DOI: 10.1021/acsami.6b00284 ACS Appl. Mater. Interfaces 2016, 8, 8947−8955
Research Article
ACS Applied Materials & Interfaces
cells. Overall, our findings indicate that NCTP can be used as an efficient carrier for drug delivery.
free-DOX.29 DOX bound onto NCTP may penetrate cells, affecting the cell cycle and resulting in a greater reduction in the expression of p-mTOR than free DOX. The Rb protein represses transcription of the gene required for transition from the G1 to the S phase and its important mediator for the activation of p21. NCTP-DOX increases the amount of Rb protein expression for an even longer period, resulting in an increased amount of p21 expression.30 This p21 is responsible for cell arrest in the G1 and G2 cell phase and known to be an important marker protein of cellular senescence.31 β-actin has frequently been used as a loading control and housekeeping gene.32 Our results showed that NCTP efficiently delivered the anticancer agent to induce apoptosis and senescence in cancer cells. Recently, Pan et al.33 synthesized poly(methacrylic acid) based nanohydogels for anticancer drug delivery. The drug (DOX) loading onto the hydrogel was up to 42.3%. The cumulative release rate of the DOX-loaded hydrogel was 91% in 5 h at pH 5.0, exhibiting a controlled drug release capability. Louquet et al.34 synthesized poly(ethylene oxide-co-propylene oxide)-b-poly(L-lysine) grafted magnetic particles for drug delivery and exhibited a 48% release of DOX at 43 °C for a period of around 8 h. Ahmady et al.35 reported the encapsulation of DOX into liposome-peptide (600:1) nanohybrid with DOX loading of 90%. The drug release rate was observed to be 70% in 24 h at 37 °C. Panday et al.36 found the drug release rate of DOX from a gold nanorods−cysteamine− folic acid−DOX complex to be 62, 52, and 62% of the DOX release at pH 5.3, 6.8, and 7.2, respectively, for 4 h at 37 °C. Wang et al.37 prepared composite material composed of iron nanoparticles decorated graphene functionalized with chitosan for drug delivery. At pH 5.1, approximately 80% of the DOX was released (72 h), whereas only 45% was released at pH 7.4, demonstrating pH-induced drug delivery from composite particles. Li et al.38 described a poly(ethylene glycol)-bpoly(L-glutamic acid)-based drug delivery system loaded with DOX. After 60 h of incubation, 72.8 and 26.4% of DOX was released at pH 5.5 and 7.4, respectively. However, some of the above systems have limitations such as fast drug release, complex procedures for materials synthesis and low drug loading efficiency. Hence, in the present work, NCTP had several advantages including biocompatibility, photosensitivity, pH response, porous structure, high electron density, easy synthesis, and high thermal and chemical stability, which could induce sustainable DOX release into cancer and normal cells, leading to effective killing of cells.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00284. (Table S1) Elemental analysis of the CTP & NCTP Elements; (Table S2) Zeta potential data of the NCTP and NCTP-DOX at different time intervals; (Scheme S1) The strategy employed to synthesize CTP; (Figure S1) (a) XRD and (b) TGA analysis of CTP; (Figure S2) Zeta potential of CTP at pH 7; (Figure S3) DLS analysis of the NCTP; (Figure S4) (a) TEM and (b) DLS of the NCTP-DOX complex; (Figure S5) (a) fluorescence and (b) excitation spectra of the DOX and NCTP-DOX; (Figure S6) Fluorescence spectra of NCTP-DOX (37°C, pH 7.4) at different time intervals; and (Figure S7) optical, confocal, and overlay images of COS-7 cells incubated with NCTP, NCTP-DOX, and DOX for 24 h (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*Fax: +82-32-872-4046. E-mail:
[email protected] (W.-S.A). *Fax: +82-32-872-4046. E-mail:
[email protected] (Y.S.H). Author Contributions ⊥
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support received from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. NRF-2014R1A2A1A11053567) and (Grant number: 2015042434) for Basic Science Research Program.
■
REFERENCES
(1) Stewart, B. W.; Weihues, P. World Cancer Report: World Health Organization, (http://www.iarc.fr/en/publications/pdfs-online/wcr/ 2003/), 2003. (2) Sinha, R.; Kim, G. J.; Nie, S.; Shin, D. M. Nanotechnology in cancer therapeutics: Bioconjugated nanoparticles for drug delivery. Mol. Cancer Ther. 2006, 5, 1909−1917. (3) Wang, J.; Huang, L.; Yang, R.; Zhang, Z.; Wu, J.; Gao, Y.; Wang, Q.; O’Hareb, D.; Zhong, Z. Recent advances in solid sorbents for CO2 capture and new development Trends. Energy Environ. Sci. 2014, 7, 3478−3518. (4) Diaz, E.; Munoz, E.; Vega, A.; Ordonez, S. Enhancement of the CO2 retention capacity of Y-Zeolites by Na and Cs treatments: Effect of adsorption temperature and water treatment. Ind. Eng. Chem. Res. 2008, 47, 412−418. (5) Liu, J.; Thallapally, P. K.; McGrail, B. P.; Brown, D. R.; Liu, J. Progress in adsorption-based CO2 capture by metal−organic framework. Chem. Soc. Rev. 2012, 41, 2308−2322. (6) Zhang, X.; Lu, J.; Zhang, J. Porosity enhancement of carbazolic porous organic frameworks using dendritic building blocks for gas storage and separation. Chem. Mater. 2014, 26, 4023−4029. (7) Ide, Y.; Kagawa, N.; Sadakane, M.; Sano, T. Precisely designed layered silicate as an effective and highly selective CO2 adsorbent. Chem. Commun. 2013, 49, 9027−9029.
■
CONCLUSIONS In summary, we successfully synthesized NCTP and used it as both a potential photosensitizer and pH-responsive nanocarrier for drug delivery. Evaluation of cytotoxicity of NCTP using COS-7 and HeLa cells revealed no significant loss of cell viability. Of particular significance, the NCTP−DOX complex was found to have a DOX-loading capacity of 200 mg/g because of its high specific surface area, π−π stacking, and hydrophobic interaction. UV−vis spectra revealed that ∼60 and ∼80% of the DOX was released from NCTP at pH 7.4 and 4.8, respectively, over 48 h. The NCTP-DOX exhibited a higher cytotoxic effect against cancer cells than free DOX, which was confirmed by bio-TEM analysis. In addition, the senescence of NCTP−DOX in HeLa cells showed that NCTP efficiently delivered the anticancer agent to induce senescence in cancer 8954
DOI: 10.1021/acsami.6b00284 ACS Appl. Mater. Interfaces 2016, 8, 8947−8955
Research Article
ACS Applied Materials & Interfaces
(28) Fiandra, L.; Colombo, M.; Mazzucchelli, S.; Truffi, M.; Santini, B.; Allevi, R.; Nebuloni, M.; Capetti, A.; Rizzardini, G.; Prosperi, D.; Corsi, F. Nanoformulation of antiretroviral drugs enhances their penetration across the blood brain barrier in mice. Nanomedicine 2015, 11, 1387−1397. (29) Schug, T. T. mTOR favors senescence over quiescence in p53arrested cells. Aging 2010, 2, 327−328. (30) Sharma, B.; Ma, W.; Adjei, I. M.; Panyam, J.; Dimitrijevic, S.; Labhasetwar, V. Nanoparticle-mediated p53 gene therapy for tumor inhibition. Drug Delivery Transl. Res. 2011, 1, 43−52. (31) Chang, B.-D.; Watanabe, K.; Broude, E. V.; Fang, J.; Poole, J. C.; Kalinichenko, T. V.; Roninson, I. B. Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: Implications for carcinogenesis, senescence, and age-related diseases. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 4291− 4296. (32) Khan, S. A.; Tyagi, M.; Sharma, A. K.; Barreto, S. G.; Sirohi, B.; Ramadwar, M.; Shrikhande, S. V.; Gupta, S. Cell-type specificity of βactin expression and its clinicopathological correlation in gastric adenocarcinoma. World J. Gastroenterol 2014, 20, 12202−12211. (33) Pan, Y.-J.; Chen, Y.-Y.; Wang, D.-R.; Wei, C.; Guo, J.; Lu, D.-R.; Chu, C.-C.; Wang, C.-C. Redox/pH dual stimuli-responsive biodegradable nanohydrogels with varying responses to dithiothreitol and glutathione for controlled drug release. Biomaterials 2012, 33, 6570−6579. (34) Louguet, S.; Rousseau, B.; Epherre, R.; Guidolin, N.; Goglio, G.; Mornet, S.; Duguet, E.; Lecommandoux, S.; Schatz, C. Thermoresponsive polymer brush-functionalized magnetic Manganite nanoparticles for remotely triggered drug release. Polym. Chem. 2012, 3, 1408−1417. (35) Al-Ahmady, Z. S.; Al-Jamal, W. T.; Bossche, J. V.; Bui, T. T.; Drake, A. F.; Mason, A. J.; Kostarelos, K. Lipid−peptide vesicle nanoscale hybrids for triggered drug release by mild hyperthermia in Vitro and in Vivo. ACS Nano 2012, 6, 9335−9346. (36) Pandey, S.; Shah, R.; Mewada, A.; Thakur, M.; Oza, G.; Sharon, M. Gold nanorods mediated controlled release of doxorubicin: nanoneedles for efficient drug delivery. J. Mater. Sci.: Mater. Med. 2013, 24, 1671−1681. (37) Wang, C.; Ravi, S.; Garapati, U. S.; Das, M.; Howell, M.; Mallela, J.; Alwarappan, S.; Mohapatr, S. S.; Mohapatr, S. Multifunctional chitosan magnetic-graphene (CMG) nanoparticles: a theranostic platform for tumortargeted co-delivery of drugs, genes and MRI contrast agents. J. Mater. Chem. B 2013, 1, 4396−4405. (38) Li, M.; Song, W.; Tang, Z.; Lv, S.; Lin, L.; Sun, H.; Li, Q.; Yang, Y.; Hong, H.; Chen, X. Nanoscaled poly(l-glutamic acid)/doxorubicinamphiphile complex as ph-responsive drug delivery system for effective treatment of nonsmall cell lung cancer. ACS Appl. Mater. Interfaces 2013, 5, 1781−1792.
(8) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an emerging platform for cancer therapy. Nat. Nanotechnol. 2007, 2, 751−760. (9) Couvreur, P.; Gref, R.; Andrieux, K.; Malvy, C. Nanotechnology for drug delivery: Applications to cancer and autoimmune diseases. Prog. Solid State Chem. 2006, 34, 231−235. (10) Liu, J.-Q.; Wu, J.; Jia, Z.-B.; Chen, H.-L.; Li, Q.-L.; Sakiyama, H.; Soares, T.; Fei, R.; Daiguebonne, C.; Guillou, O.; Ng, S. W. Two isoreticular metal−organic frameworks with CdSO4-like topology: selective gas sorption and drug delivery. Dalton Trans. 2014, 43, 17265−17273. (11) Horcajada, P.; Chalati, T.; Serre, C.; Gillet, B.; Sebrie, C.; Baati, T.; Eubank, J. F.; Heurtaux, D.; Clayette, P.; Kreuz, C.; Chang, J.-S.; Hwang, Y. K.; Marsaud, V.; Bories, P.-N.; Cynober, L.; Gil, S.; Férey, G.; Couvreur, P.; Gref, R. Porous metal−organic framework nanoscale carriers as a potential platform for drug delivery and imaging. Nat. Mater. 2010, 9, 172−178. (12) Zhuang, J.; Kuo, C.-H.; Chou, L.-Y.; Liu, D.-Y.; Weerapana, E.; Tsung, C.-K. Optimized metal-organic-framework nanospheres for drug delivery: evaluation of small−molecule encapsulation. ACS Nano 2014, 8, 2812−2819. (13) Wang, J.-J.; Hu, T.-L.; Bu, X.-H. Cadmium (II) and zinc (II) metal−organic frameworks with anthracene-based dicarboxylic ligands: solvothermal synthesis, crystal structures, and luminescent properties. CrystEngComm 2011, 13, 5152−5161. (14) Tanmoy, M.; Debraj, S.; Soma, D.; Subratanath, K. Barium carboxylate metal−organic framework -Synthesis, X-ray crystal structure, photoluminescence and catalytic study. Eur. J. Inorg. Chem. 2012, 30, 4914−4920. (15) Zou, X.; Ren, H.; Zhu, G. Topology-directed design of porous organic frameworks and their advanced applications. Chem. Commun. 2013, 49, 3925−3936. (16) Zhao, H.; Jin, Z.; Su, H.; Jing, X.; Sun, F.; Zhu, G. Targeted synthesis of a 2D ordered porous organic framework for drug release. Chem. Commun. 2011, 47, 6389−6391. (17) Fornari, F. A.; Randolph, J. K.; Yalowich, J. C.; Ritke, M. K.; Gewirtz, D. A. Interference by doxorubicin with DNA unwinding in MCF-7 breast-tumor cells. Mol. Pharmacol. 1994, 45, 649−656. (18) Momparler, R. L.; Karon, M.; Siegel, S. E.; Avila, F. Effect of adriamycin on DNA, RNA, and protein-synthesis in cell-free systems and intact-cells. Cancer Res. 1976, 36, 2891−2895. (19) Artandi, S. E.; DePinho, R. A. A critical role for telomeres in suppressing and facilitating carcinogenesis. Curr. Opin. Genet. Dev. 2000, 10, 39−46. (20) De Lange, T.; Jacks, T. For better or worse? Telomerase inhibition and cancer. Cell 1999, 98, 273−275. (21) Ben-Porath, I.; Weinberg, R. A. The signals and pathways activating cellular senescence. Int. J. Biochem. Cell Biol. 2005, 37, 961− 976. (22) Lim, H.; Cha, M. C.; Chang, J. Y. Preparation of microporous polymers based on 1,3,5-triazine units showing high CO2 adsorption capacity. Macromol. Chem. Phys. 2012, 213, 1385−1390. (23) Ki-Won, K.; Ha-Na, C.; Kee-Yong, H.; Jun-Seok, L.; Young-Yul, K. Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs. Spine Journal 2009, 9, 658−666. (24) Shijie, R.; Michael, J.; Bojdys, R.; Andrea, L.; Yaroslav, Z.; Khimyak, D.; Adams, J.; Andrew, I. Porous, fluorescent, covalent triazine-based frameworks via room-temperature and microwaveassisted synthesis. Adv. Mater. 2012, 24, 2357−2361. (25) Xiang, Z.; Cao, D. Synthesis of Luminescent Covalent−Organic Polymers for Detecting Nitroaromatic Explosives and Small Organic Molecules. Macromol. Rapid Commun. 2012, 33, 1184−1190. (26) Liu, J.; Zong, E.; Fu, H.; Zheng, S.; Xu, Z.; Zhu, D. Adsorption of aromatic compounds on porous covalent triazine-based framework. J. Colloid Interface Sci. 2012, 372, 99−107. (27) Yin, W.; Tian, G.; Ren, W.; Yan, L.; Jin, S.; Gu, Z.; Zhou, L.; Li, J.; Zhao, Y. Design of multifunctional alkali ion doped CaF2 upconversion nanoparticles for simultaneous bioimaging and therapy. Dalton Trans. 2014, 43, 3861−3870. 8955
DOI: 10.1021/acsami.6b00284 ACS Appl. Mater. Interfaces 2016, 8, 8947−8955
