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Self-Delivery Nanoparticles of Amphiphilic MethotrexateGemcitabine Prodrug for Synergistic Combination Chemotherapy via Effect of Deoxyribonucleotide Pools Yao Wang, Ping Huang, Minxi Hu, Wei Huang, Xinyuan Zhu, and Deyue Yan Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00503 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 14, 2016

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Bioconjugate Chemistry

Self-Delivery

Nanoparticles

of

Amphiphilic

Methotrexate-Gemcitabine Prodrug for Synergistic Combination

Chemotherapy

via

Effect

of

Deoxyribonucleotide Pools

Yao Wang, Ping Huang, Minxi Hu, Wei Huang, Xinyuan Zhu,* and Deyue Yan*

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China

* Corresponding authors. E-mail: [email protected] (X.Z.); [email protected] (D.Y.). Telephone: +86-21-54746215. Fax: +86-21-54741297.

ACS Paragon Plus Environment


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ABSTRACT: The distinct and complementary biochemical mechanisms of folic acid analog methotrexate (MTX) and cytidine analog gemcitabine (GEM) make their synergistic combination effectively. Unfortunately, such a combination faces severe pharmacokinetic problems and several transportation barriers. To overcome these problems, a new strategy of amphiphilic small molecule prodrug (ASMP) is developed to improve their synergistic combination effect. The ASMP was prepared by the amidation of the hydrophilic GEM with the hydrophobic MTX at a fixed ratio. Owing to its inherent amphiphilicity, the MTX-GEM ASMP self-assembled into stable nanoparticles (ASMP-NPs) with high drug loading capacity (100%), in which the MTX and GEM could self-deliver without any carriers and release synchronously in cancer cells. In vitro studies showed that the MTX-GEM ASMP-NPs could greatly improve the synergistic combination effects by the reason of arresting more S phase of the cell cycle and reducing more levels of deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP). The stronger synergistic effects caused the higher cell cytotoxicity and apoptotic ratio, and circumvented the multidrug resistance (MDR) of tumor cells. Additionally, MTX-GEM ASMP-NPs could achieve the same anticancer effect with the greatly lower dosage comparing with the free drugs according to the dose-reduction index (DRI) values of MTX and GEM in MTX-GEM ASMP-NPs, which may be beneficial to reduce the side effects.


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INTRODUCTION Antimetabolites are the most widely used and efficacious group of anticancer drugs. Each antimetabolite often has multiple targets and it differs from one another in their mechanism of actions, leading to different cytotoxic effects. Therefore, their complementary anticancer mechanisms endow antimetabolites with wide usages in combination chemotherapy for treatment of various leukemias and solid tumors. MTX and GEM are both known to act as one of the most extensively used antimetabolites during the therapy of head and neck cancer, breast cancer and some other solid tumors.1-3 They inhibit different steps in the synthesis of DNA and RNA. The main mechanism of GEM activation is most likely due to the conversion to nucleotides gemcitabine triphosphate (GEM-dCTP) as an inhibitor of DNA polymerase.4 GEM-dCTP could also incorporate into DNA and RNA,5 leading to termination of chain elongation and breakage of single strand.6 The above molecular process is essential to the apoptosis of GEM. MTX is an inhibitor of dihydrofolate reductase (DHFR). The DHFR’s product tetrahydrofolates (THF) and the closely related thymidylate synthase (TS) substrate 5,10-methylene tetrahydrofolate (CH2THF) could be exhausted adequately in the presence of MTX to cause inhibition of the synthesis of purines and thymidylate.7 Therefore, MTX interrupts the synthesis of DNA and RNA, similar to GEM. Notably, MTX and GEM have distinct but complementary biochemical mechanisms to reach the same goal. As a result, using a combination of MTX and GEM could result in enhanced inhibition of the synthesis of DNA with different mechanisms of action. Because of their superior synergy, the combination of



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MTX and GEM has already been effectively applied to primary clinical chemotherapy for the treatment of relapsing head and neck cancer and malignant pleural mesothelioma.8,9 Unfortunately, it has reported that both MTX and GEM have severe pharmacokinetic problems during the treatment of cancer. GEM possesses rapid body clearance with short plasma half-life that limits its efficacy. Furthermore, GEM is inactivated and converted to difluorodeoxyuridine (dFdU) mainly by deoxycytidine deaminase (dCDA) predominantly in the liver, plasma and peripheral tissues.10 In addition, anticancer efficacy of MTX is also severely reduced by short bloodstream half-life, dose-related side effects, and the development of resistance of cancer cells. The performances of resistance to MTX decrease the drug influx, increase the hydrolysis of MTX, and so on.10 Consequently, frequent administrations at high drug dosage are needed, which result in the higher levels of side effects.11,12 To address the aforementioned limitations, Jain and coworkers constructed a novel macromolecular bipill of GEM and MTX which were linked to the two terminals of poly(ethylene glycol) (PEG) respectively.13 Importantly, the macromolecular bipill based delivery systems could remarkably improve the solubility and stability of constituent drug molecules in plasma. The PEG carrier was indispensable in the macromolecular bipill delivery systems, which leads to a low drug loading capacity. Even worse, the drawbacks of PEG are of major concern to us in these days: hypersensitivity, toxic side products, and its non-biodegradability, as well as the resulting possible accumulation in the body.14 Provided that MTX and GEM could


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deliver themselves without the help of carriers whilst integrating both the advantages of free drugs and nanodelivery systems, a promising drug co-delivery system for MTX and GEM could be expected. To achieve this goal, herein we designed and synthesized an amphiphilic small molecule prodrug (ASMP) to improve the synergistic effect of MTX and GEM (Figure 1), in which the hydrophobic anticancer drug MTX and the hydrophilic anticancer drug GEM were linked each other with the amide bond (1:1). The dosage ratio of MTX and GEM is always fixed when the degradation of ASMP is induced in cancer cells, resulting in the maximization of combinatorial effects. Owing to its amphiphilicity, the MTX-GEM ASMP could self-assemble into nanoparticles (MTX-GEM ASMP-NPs) in aqueous solution, and then deliver themselves effectively into tumor tissues without help of any carriers.15,16 After the cellular internalization of MTX-GEM ASMP-NPs, both native MTX and GEM could be released simultaneously at fixed ratio to kill the cancer cells, resulting from the hydrolysis of the amide bond in the tumor cells. The anticancer effect was evaluated in vitro and the mechanisms of the synergistic combination were explored. The results demonstrate that the carrier-free MTX-GEM ASMP-NPs hold the potential to be further engineered as combinatorial drug delivery system with great synergistic therapeutic function.



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Figure 1. Schematic route of MTX-GEM ASMP and construction of self-assembled nanoparticles (MTX-GEM ASMP-NPs) for cancer combination chemotherapy.

RESULTS AND DISCUSSION Synthesis and Characterization of MTX-GEM ASMP. The synthetic route of the MTX-GEM ASMP is shown in Figure 1. In order to obtain the MTX-GEM ASMP, MTX is linked with GEM by amide with a molar feed ratio of MTX/GEM at 1:1.05 to produce the amphiphilic MTX-GEM ASMP. Typically, MTX improves the protection against deamination of GEM’s amino via the formation of amide bond and then it supplements a beneficial implement to improve the stability of GEM against rapid plasma inactivation.17,18


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As described in Figure 2B,C by 1H NMR and 13C NMR spectroscopy, the chemical structure identification data of MTX-GEM ASMP were determined. For the 1H NMR, the integral area of the amino proton signal at 7.36 ppm in MTX-GEM ASMP is half of that of the amide proton signal at 7.64 ppm in GEM. Moreover, the two hydroxyl proton signals at 5.76 ppm and 5.18 ppm belonging to GEM show their existence at 6.41 ppm and 5.35 ppm in MTX-GEM ASMP, respectively. Therefore, MTX is linked with GEM only at the position of -NH2 group in GEM, as further proved by the shifting of H2’ in GEM signals from 6.21 ppm to 7.24 ppm in MTX-GEM ASMP. The peak at 2.22 ppm (H3) related to -CH2- of MTX shifts to 2.30 ppm in MTX-GEM ASMP, which gives a proof that -CH2-CH2-COOH of MTX is reacted with the -NH2 group of GEM. The same conclusion has also been testified by 13C NMR. In Figure 2C, the 174.51 ppm signal ascribing to -COOH (C1) of MTX shifts to 176.87 ppm in MTX-GEM ASMP. The signal at 155.52 ppm corresponding to NH2-C (C1’) and the signal at 95.13 ppm attributed to C2’ in GEM shift to 160.01 ppm and 96.41 ppm in MTX-GEM ASMP, respectively. These data further confirm the acylation. Furthermore, the 68.56 ppm (C4’) and 59.09 ppm (C5’) signals from the methyne (-CH-OH) and the methylene (-CH2OH) of GEM remain unchanged. To verify the purity of MTX-GEM ASMP, we performed liquid chromatography (LC) and mass spectrometry (MS) measurements and the results are given in Figure 2D. Only one retention time at 3.61 min is observed in the LC profile of the MTX-GEM ASMP, which demonstrates the high purity of MTX-GEM ASMP. In addition, the MS result confirms that the molecular weight of MTX-GEM ASMP (m/z, M-H+) is 698.2197, which is consistent with the calculated



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value (m/z, M-H+, 698.2247). The relatively high peak at 435 in Fig. 2d is the fragment peak of MTX-GEM ASMP which comes from the cleavage of carbon-nitrogen bond (OC-NH) in the new generated amide which links MTX and GEM. These results demonstrate that the MTX-GEM ASMP has been synthesized successfully. The MTXGEM ASMP was further determined by fluorescence spectroscopy and ultravioletvisible (UV-Vis) spectrophotometer, and the results are exhibited in Figure S1 and S2, respectively. Owing to the fluorescence of MTX at 440 nm, the MTX-GEM ASMP presents a fluorescence emission at 437.5 nm. In addition, as depicted in Figure S2, both GEM and MTX absorptions can be observed in the UV-Vis spectrum of MTXGEM ASMP. Furthermore, a 2 nm blue-shift in the UV-Vis absorption of MTX-GEM ASMP at 381 nm is observed, by contrast with the absorption of MTX at 383 nm. We also observe a distinct reinforcement at 266 nm in the absorption of the MTX-GEM ASMP, resulting from the introduced structure of GEM. These experimental results further demonstrate the successful synthesis of MTX-GEM ASMP.


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Figure 2. (A) Structure of MTX-GEM ASMP. (B) 1H NMR and (C) 13C NMR spectra of MTX, GEM and MTX-GEM ASMP in dimethyl sulfoxide-d6 (DMSO-d6). (D) MS of MTX-GEM ASMP. Inset: the LC profile of MTX-GEM ASMP. (E) The relationship of fluorescence intensity of Nile red (NR) and the MTX-GEM ASMP-NPs concentrations. The CAC value of MTX-GEM ASMP-NPs is about 7.06 μM. (F) Dynamic light scattering (DLS) plot of MTX-GEM ASMP-NPs, which shows the average size (Dh = 206.2 nm) and the polydispersity index (PDI = 0.132). (G) Transmission electron microscopy (TEM) image of MTX-GEM ASMP-NPs. (H) In vitro GEM release kinetics from MTX-GEM ASMP-NPs under different pH values (5.0 and 7.4) containing 25% fetal bovine serum (FBS) (or not) at 37 °C. Fabrication and Characterization of Self-Assembled MTX-GEM ASMP-NPs. The MTX-GEM ASMP could self-assemble into nanoparticles in water resulting from the hydrophobicity of MTX and hydrophilicity of GEM. The self-assembled MTXGEM ASMP-NPs were obtained by the dialysis method. The N,N-dimethylformamide (DMF) solution of MTX-GEM ASMP was added dropwise into the deionized water. After that, a stable MTX-GEM ASMP-NPs solution (0.5 mg mL-1) was achieved through removing DMF by dialysis. To validate whether MTX-GEM ASMP could self-assemble into nanoparticles in aqueous solution or not and evaluate the self-assembly behavior of MTX-GEM ASMPNPs in aqueous solution, the critical aggregation concentration (CAC) of MTX-GEM ASMP-NPs was investigated using Nile red (NR) as the fluorescence probe. As displayed in Figure 2E, when the concentration of MTX-GEM ASMP-NPs is low, the


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fluorescence emission intensity of NR raises tardily. Nevertheless, when a certain concentration is surpassed, the fluorescence intensity increases remarkably which implies that the micelles begin to form and NR has been encapsulated into the hydrophobic cores. The specific concentration could be defined as CAC which is procured as the intersection of the tangents to the two linear portions of the graph. It is suggested that the CAC of MTX-GEM ASMP in aqueous solution is about 7.06 μM. The low CAC of MTX-GEM ASMP is beneficial to promote the formation of nanoparticles. To further determine the characteristic size and morphology of MTX-GEM ASMPNPs, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were performed. The DLS analysis in Figure 2F indicates the formation of aggregates with a narrow monomodal distribution (polydispersity index, PDI: 0.132) and an average hydrodynamic diameter of approximate 206.2 nm. As showed in Figure 2G, the morphology of the MTX-GEM ASMP-NPs visualized by TEM is approximately spherical micelles with an average diameter of 141.3 nm (n = 100). Obviously, the diameter of the micelles determined by DLS is a little larger than that visualized in TEM. Such a difference is reasonable owing to the hydration of the shell portion of the micelles.19 In Vitro Release Behavior from MTX-GEM ASMP-NPs. The dialysis method is applied to investigate release behavior in vitro of MTX-GEM ASMP-NPs. The simulated physiological condition (phosphate-buffered saline, PBS, pH = 7.4) containing (or not) 25% FBS and acidic ambient (acetate buffer, pH = 5.0) containing



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(or not) 25% FBS at 37 °C were studied. As displayed in Figure 2H, the cumulative release curves present that release behavior in vitro of MTX-GEM ASMP-NPs is a process of slow and sustained release. As shown in Figure 2H, there is only a slow release of about 8% and 10% of GEM respectively in pH = 7.4 PBS solution containing (or not) 25% FBS within a period of 26 h, which demonstrates that the MTX-GEM ASMP-NPs exhibit high stability in the physiological condition. However, more free GEM drugs (20%) are released at a weakly acidic environment (pH = 5.0). Especially, comparing with other conditions, the MTX-GEM ASMP-NPs release 25% of GEM in a weakly acidic environment (pH = 5.0) containing 25% FBS within 26 h. because the 25% FBS and acidic condition could accelerate the degradation of amide bond.13,20 The effect of 25% FBS may result from a series of esterases, peptidases or other enzymes in serum.21,22 To confirm whether the MTX-GEM ASMP was indeed degraded within the tumor cells, we conducted a study of the intracellular degradation. After the cell incubation with MTX-GEM ASMP-NPs for 24 h at 37 °C, we took liquid chromatography-mass spectrometry (LC-MS) technique to analyze the cellular extracts of the CAL-27 cells (human squamous tongue cancer cell line). The LC-MS data in Figures S3 and S4 show the peaks of MTX and GEM. The results present that the amide bond between MTX and GEM is indeed cleaved in cells and then two original active principles are released in cancer cells. Cell Internalization. Whether the MTX-GEM ASMP-NPs could effectively and quickly transport into cancer cells is a crucial aspect for therapeutic efficacy. We


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analyze the cellular uptake of MTX-GEM ASMP-NPs in CAL-27 cell line through the flow cytometry and laser confocal scanning microscopy (CLSM) technique. The NRloaded MTX-GEM ASMP-NPs solution was prepared for the experiments. For analysis of the flow cytometry, the CAL-27 cells were incubated with the NR-loaded MTXGEM ASMP-NPs at 37 °C for the predetermined time intervals (15 min, 0.5 h, 1 h, 2 h and 4 h). The cells untreated with the NR-loaded MTX-GEM ASMP-NPs solution were taken as a control. The fluorescence intensity of NR in CAL-27 cells gradually strengthens with the extension of culture duration (Figure 3A). The relative geometrical mean fluorescence intensity of the non-pretreated cells is only 166. However, the relative geometrical mean fluorescence intensity of the cells pretreated by NR-loaded MTX-GEM ASMP-NPs for 4 h is 8772, which is 53-fold of that of non-pretreated cells (Figure 3B). The distinct escalation of fluorescence intensity displays that the NRloaded MTX-GEM ASMP-NPs could be efficiently internalized by CAL-27 cells and the MTX-GEM ASMP-NPs exhibit effective cellular uptake.



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Figure 3. Cellular uptake of MTX-GEM ASMP-NPs by CAL-27 cells. (A) The relationship of fluorescence intensity and time of NR-loaded MTX-GEM ASMP-NPs


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in the CAL-27 cells by flow cytometry analysis. (B) Flow cytometry histogram profiles of CAL-27 cells that were incubated with NR-loaded MTX-GEM ASMP-NPs for 0 h (control) and 4 h. (C) The CLSM images of CAL-27 cells incubated with NR-loaded MTX-GEM ASMP-NPs for 15 min, 0.5 h, 1 h, 2 h and 4 h. Cell nuclei were stained with YOYO-1. Moreover, the cellular uptake of MTX-GEM ASMP-NPs was further evaluated by CLSM. CAL-27 cells were cultured with NR-loaded MTX-GEM ASMP-NPs at 37 °C for 15 min, 0.5 h, 1 h, 2 h and 4 h before observation, and the nuclei were stained by YOYO-1 for 15 min. Thereafter, the location of drugs in cells was analyzed by taking advantage of the red fluorescence from NR and the green fluorescence from YOYO-1. As displayed in Figure 3C, the fluorescence intensity of NR in the cells preprocessed with NR-loaded MTX-GEM ASMP-NPs for 15 min is weak in cytoplasm. The intensity of fluorescence is strengthened gradually when the incubation time is extended from 0.5 h to 4 h. Consequently, the effective internalization of MTX-GEM ASMP-NPs by cancer cells is validated. In Vitro Cytotoxicity of MTX-GEM ASMP-NPs. The proliferation inhibition of MTX-GEM ASMP-NPs comparing with free MTX, GEM and MTX+GEM mixture was assessed against CAL-27 and MCF-7 cells (human breast adenocarcinoma cell line) under identical conditions by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We prepared a series of nanoparticles and free drug concentrations to treat the cells. The untreated cells were taken as a control. The results of cell proliferation are given in Figure 4A,B. Obviously, compared to the other three



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drug formulations (the half maximal inhibitory concentration, IC50, GEM = 1.99 μM, IC50, MTX

= 8.51 μM, IC50, MTX/GEM = 0.90 μM), MTX-GEM ASMP-NPs exhibit significant

inhibition to the cell proliferation (IC50 = 0.46 μM), particularly at the higher concentration range above CAC value after incubation for 72 h. Also, the same results appear in the MCF-7 cells (Figure 4B). Synergistic Effect. Strong synergism in therapeutic efficacy (combination index, CI < 0.4) and significant dose reduction (DRI > 1) for a given effect are recognized as the best scenarios for two drug combination.23 To study the synergy of MTX and GEM in MTX-GEM ASMP-NPs, the CI values, which can present the drug interaction nature qualitatively, are calculated by the Chou-Talalay equation.24,25 As shown in Figure 4C,D, all of the CI values of MTX+GEM mixture and MTX-GEM ASMP-NPs with 72 h incubation are below the line of CI = 1. In addition, the CI values of MTX-GEM ASMP-NPs are much lower than those of MTX+GEM mixture at each corresponding ICx value, denoting that the synergistic effect of MTX-GEM ASMP-NPs is more remarkable than that of the mixture of two free drugs. Notably, most of the CI values of MTX-GEM ASMP-NPs are below 0.4, which refers to the strong synergy. Furthermore, another major objective of synergistic drug combination is to reduce the dosage of the single drug used alone while maintaining its anticancer efficacy. DRI indicates the folds of dose-reduction in a synergistic combination at a given effect level (DRIx), in contrast to the single drug used alone.23 The DRIx-DRI plot provides the DRI values of each drug at different effect levels by computer simulation.23 In Figure 4E and 4F, all of the DRI values of MTX+GEM mixture and MTX-GEM ASMP-NPs


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are above the line of DRI = 1 with 72 h incubation of drugs. Additionally, compared to MTX+GEM mixture, the DRI values of MTX and GEM in MTX-GEM ASMP-NPs are very high, especially at larger concentrations. As shown in Figure 4E, the DRI values of MTX and GEM in MTX-GEM ASMP-NPs at inhibition of 50% are 18.45 and 4.32 in CAL-27 cells, which are almost 2-fold of those in MTX+GEM mixture (9.42 and 2.22). Likewise to MCF-7 cells, the DRI values of MTX and GEM in MTX-GEM ASMP-NPs at inhibition of 50% are 9.00 and 7.30, whereas those in MTX+GEM mixture are 3.814 and 3.096 respectively. Thus, the use of MTX-GEM ASMP-NPs could largely reduce the dosage of free drugs while maintaining effective anticancer efficacy.



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Figure 4. Cell viability of (A) CAL-27 cells and (B) MCF-7 cells incubated with MTX, GEM, MTX+GEM mixture, and MTX-GEM ASMP-NPs after 72 h at various concentrations determined by MTT assay. CI of MTX and GEM combinations via


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different formulations effects on (C) CAL-27 cells and (D) MCF-7 cells. Synergism is subdivided into slight synergism (+), moderate synergism (++), synergism (+++), strong synergism (++++), and very strong synergism (+++++). The DRI values of MTX+GEM mixture and MTX-GEM ASMP-NPs on (E) CAL-27 cells and (F) MCF7 cells. Values are presented as average  standard error (n = 3). (G) Caspase-3 protein activity in CAL-27 cells activated by MTX, GEM, MTX+GEM mixture and MDR at a concentration of 35 M. Significantly different from control levels with *P < 0.05, **P < 0.01 and ***P < 0.001. (H) Cell viability of MCF-7/ADR cultured with MTX, GEM, MTX+GEM mixture, and MTX-GEM ASMP-NPs determined by MTT assay after 72 h. (I) Cell apoptosis data of CAL-27 cells treated with GEM, MTX, MTX+GEM mixture and MTX-GEM ASMP-NPs for 48 h by flow cytometry analysis. Inserted numbers in the profiles present the percentage of the cells in this area. Lower left: living cells; Upper left: necrotic cells; Lower right: early apoptotic cells; Upper right: late apoptotic cells. Multidrug Resistance. The MDR of tumor cells has been a considerable intervention to the success of cancer chemotherapy, particularly small-molecule drugs. One of the current effective strategies to reverse the tumor MDR refers to nanotechnology,26 which could facilitate the small molecule anticancer drugs bypassing the P-gp efflux pump and promote their accumulation in tumors cells.27 Hence, it is expected that MTX-GEM ASMP-NPs would have the potential to retrograde the MDR of tumor cells. To validate whether the MTX-GEM ASMP-NPs would show higher cytotoxicity on drug-resistant MDR tumor cells comparing with single drug, we took



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MTT assay to evaluate the cytotoxicity of GEM, MTX, MTX+GEM mixture and MTXGEM ASMP-NPs on MCF-7/ADR cells (Figure 4H) and IC50 values were calculated simultaneously (Table S1). As shown in Figure 4H, by reason of the overexpression of P-gp, MCF-7/ADR cells show a high tolerance when treated with MTX, GEM and MTX+GEM mixture, as compared to that of its counterpart drug-sensitive MCF-7 cells (Figure 4B) in the MTT assays. The IC50 values of GEM and MTX+GEM mixture are 159.90 M and 45.02 M in the MCF-7/ADR cells individually and are 10.92 M and 3.52 M in MCF-7 cells. The resistance index (RI) of MTX and MTX+GEM mixture are 14.56 and 12.79, respectively (Table S1). However, the IC50 (2.76 M) of MTXGEM ASMP-NPs in MCF-7/ADR cells is just 2.76 M and the RI is only 1.86. The data demonstrate that the MTX-GEM ASMP-NPs could be applied to effectively reduce the effect of MDR. Moreover, the amphiphilic small molecule prodrug nanoparticle strategy opens up an effective avenue to overcome the MDR, resulting in an excellent anticancer activity. Cell Apoptosis Induced by MTX-GEM ASMP-NPs. Generally, cell apoptosis is a principal mechanism for most small molecular chemotherapy drugs. To confirm whether the cell apoptosis is an important factor to induce the death of cancer cells incubating with MTX-GEM ASMP-NPs, we applied FITC-Annexin V/PI apoptosis detection kit as the effective measure to qualify the ratio of apoptosis cells. GEM, MTX, MTX+GEM mixture and MTX-GEM ASMP-NPs at the same concentration of 35 M treated the CAL-27 cells for 48 h and subsequently the cells were stained by FITCAnnexin V/PI. The cells without treatment were applied as a control. As shown in


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Figure 4I, the apoptotic percentages induced by GEM, MTX, MTX+GEM mixture and MTX-GEM ASMP-NPs are 11.36%, 24.22%, 27.21%, and 53.28% respectively. The data demonstrate that MTX-GEM ASMP-NPs could induce higher apoptotic rate of CAL-27 cells comparing with other drug formulations at the same concentration. Caspase-3 Protein Activity Assay. Caspases, a family of cysteine-aspartyl proteases, is critical to signal transforming of apoptosis and inflammation. 28,29 Especially, caspase-3 is regarded as a key effector in the caspase-dependent apoptotic pathway, and it is noted being up-regulated with the activation of cytotoxic drugs. 30,31 To examine whether the MTX-GEM ASMP-NPs could activate caspase-3, the substrate of Ac-DEVD-pNA was used to quantify the expression level of caspase-3 protein. MTX, GEM, MTX+GEM mixture and MTX-GEM ASMP-NPs at the uniform concentration of 35 M treated the CAL-27 cells for 48 h. The cells untreated with drugs were used as a control. As demonstrated in Figure 4G, after the treatment with GEM, MTX, and MTX+GEM mixture in CAL-27 cells, the caspase-3 protein expression increases distinctly in comparison with the control (4.71-fold, 4.42fold, 5.29-fold of the control cells, separately). Notably, compared to the CAL-27 cells treated with GEM, MTX, MTX+GEM mixture, the expression of caspase-3 protein of the CAL-27 cells treated with MTX-GEM ASMP-NPs is up-regulated higher (5.96fold of the control cells). Therefore, although caspase-3 can be activated by GEM, MTX, MTX+GEM mixture largely, the results obviously confirm that the MTX-GEM ASMPNPs present the most efficient avenue to activate the activity of caspase-3. Effects of MTX-GEM ASMP-NPs on Cell Cycle Distribution. The effects on the



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cell cycle distribution are determined to discuss the reason of the synergistic combination of MTX and GEM and to explore whether MTX-GEM ASMP-NPs could be used to optimize the combination of the two drugs. MTX and GEM mainly arrest the cell cycle in S-phase.32,33 Moreover, the apoptotic signal triggered by MTX is likely to be initiated at the time of DNA synthesis: S phase of the cell cycle.34 Also, GEM, a cell cycle-dependent deoxycytidine analogue of the antimetabolite class, would be more effective when the majority of cells are in S phase.35 Therefore, the continually increased proportion of S phase could maximally improve the anticancer effect of GEM and MTX. It can be inferred that the combination of MTX and GEM would enhance the effects on cell cycle by increasing the population of S-phase for each other and the continually increased proportion of S phase could maximally improve the anticancer effect of GEM and MTX. As seen in Figure 5, the S-phase arrest appears to be at 23.21% and 29.46% by MTX and GEM treatment, whereas MTX+GEM mixture arrests the cells population in S-phase by 32.50%. However, the treatment of MTX-GEM ASMPNPs has a much higher influence on cell cycle progression, which produces a prominent delay in progression through S-phase by 40.87% after 48 h. Thus, the results suggest that MTX-GEM ASMP-NPs can largely enhance and optimize the synergistic combination of MTX and GEM.

Figure 5. Effects of MTX, GEM, MTX+GEM mixture and MTX-GEM ASMP-NPs


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treatments on cell cycle distribution in CAL-27 cells. Cells were treated with 35 μM of MTX, GEM, MTX+GEM mixture and MTX-GEM ASMP-NPs for 48 h. Cell cycle distribution was determined by analyzing 10000 gated cells. Content of DNA is represented on the x-axis; number of cells counted is represented on the y-axis.

Figure 6. Mechanisms of action and metabolism of MTX and GEM.7,18,36,37 GEMdCMP:

nucleotides

gemcitabine

monophosphate;

GEM-dCDP:

nucleotides

gemcitabine diphosphate; GEM-dCTP: nucleotides gemcitabine triphosphate; dCMPDA: deoxycytidine monophosphate deaminase; DHF: dihydrofolate; dFdUMP: 2′,2′-difluorodeoxyuridine monophosphate; SHMT: serine hydroxymethyltransferase. The Levels of dCTP, dTTP and dATP Following the Combination of MTXGEM ASMP-NPs. Altered dCTP, dTTP and dATP levels may play a role in the inhibition of DNA synthesis from MTX and the increased GEM incorporation into DNA.18,38 Therefore, we investigated whether changes in dCTP, dTTP and dATP levels induced by GEM and MTX result in the good synergistic combination of both drugs. As shown in Figure 6, MTX is a potent inhibitor of DHFR and gives rise to inhibition of intracellular thymidylate synthesis, and DNA synthesis. 39-41 In the presence of MTX,



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the DHFR product, THF and the closely related TS substrate, CH2THF, are sufficiently depleted to cause disruption of TS.1,42-44 Correspondingly, the level of dTTP is lowered efficiently. The antitumor activity of GEM depends on a series of sequential phosphorylations. At the first rate-limiting step, deoxycytidine kinase converts GEM to the monophosphorylated metabolite, GEM-dCMP. GEM-dCMP could be deaminated to dFdUMP by dCMPDA. Theoretically, dFdUMP may serve as either a substrate or an inhibitor of TS.18 The possible suppression of TS by GEM can induce the depletion of thymidine monophosphate (dTMP) pools45 and then the depletion of dTTP. Moreover, subsequent phosphorylations of GEM-dCMP lead the accumulation of GEM-dCDP and GEM-dCTP which are both active metabolites. However, GEMdCDP could result in the inhibition of ribonucleotide reductase and subsequently reduce the synthesis of deoxynucleoside triphosphates: dCTP, dTTP, specifically dATP (in solid tumor cells).36,37 GEM-dCTP is competitive with endogenous dCTP as both an inhibitor of and a substrate for DNA synthesis. This occurs because the phosphorylation of GEM by deoxycytidine kinase can also be inhibited by dCTP.46,47 In general, as shown in Figure 6, the combination of MTX and GEM may block the synthesis of DNA by indirectly reducing the levels of dTTP, dCTP and dATP and the increased GEM incorporation into DNA. The more decreased dTTP, dCTP and dATP levels could testify the better synergistic combination of MTX and GEM in cancer research. As illustrated in Figure 7, when the MTX+GEM mixture treat the CAL-27 cells, more levels of dTTP, dCTP and dATP are decreased comparing with single drug. It’s worth noting that the CAL-27 cells treated with MTX-GEM ASMP-NPs could reduce the


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levels of dTTP, dCTP and dATP to the minimum. The result is consistent with that of cell cycle. It is elucidated that MTX-GEM ASMP-NPs could optimize the synergistic combination of both drugs efficiently.

dNTP levels (% of control)

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120

MTX GEM MTX+GEM mixture MTX-GEM ASMP-NPs

*

100 80 60 40 20 0

** **

*** ** *** ** ** ** ** ******

dCTP

dTTP

dATP

Figure 7. Effects of MTX, GEM, MTX+GEM mixture and MTX-GEM ASMP-NPs on dCTP, dTTP and dATP pools in CAL-27 cells. Cells were treated with MTX, GEM, MTX+GEM mixture and MTX-GEM ASMP-NPs at a concentration of 35 μM for 48 h. Significantly different from control levels with *P < 0.05, **P < 0.01 and ***P < 0.001. Conclusions. In summary, we designed and prepared an amphiphilic small molecular prodrug (MTX-GEM ASMP), which could improve the synergistic combination of MTX and GEM by the reason of largely arresting the cells population in S-phase and reducing the levels of dTTP, dATP and dCTP. The MTX-GEM ASMP was endowed with high drug loading capacity (100%) and a fixed ratio of MTX and GEM. Due to the inherent amphiphilic nature, in aqueous solution, the MTX-GEM ASMP could self-assemble into nanoparticles. Profiting from the nanoscale features, therapeutic efficacy could be maximized. In comparison with the free drugs, MTX-



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GEM ASMP-NPs could achieve MDR reversal as well as result in much higher expression level of caspase-3 and apoptotic rate. Moreover, MTX-GEM ASMP-NPs could achieve the same anticancer effect with the lower dosage compared to the free drugs. Due to the benefits of the ASMP, it is anticipated that the tactic may proceed a new avenue to promote the further advancements of synergistic combination system and can found its potential use in clinic in the future. METHODS Synthesis of MTX-GEM ASMP. The detailed synthesis route of MTX-GEM ASMP is described as follows: To a solution of MTX (908.9 mg, 2.0 mmol) in 25 mL dried DMF, DCC (494.8 mg, 2.4 mmol) and DMAP (24.4 mg, 2.4 mmol) were added. Kept in the ice bath, the mixture was stirred for 0.5 h. Then, a solution of GEM·HCl (629.3 mg, 2.1 mmol) and TEA (334.7 μL, 2.4 mmol) in 6 mL dried DMF was injected dropwise. The resulting solution was stirred at room temperature for another 48 h. Subsequently, the reaction mixture was filtered to remove the white solid and the filtrate was dried under vacuum. The crude product was purified by column chromatograph using

ethyl

acetate

(C2H5COOCH3)

and

dichloromethane/methanol

(C2H5COOCH3:CH3OH, 3:4 v/v) as the eluent. The product was collected and then dried under vacuum to give a yellow solid (853.6 mg, 65%). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.54 (s, 1H), 8.25-8.23 (dd, J = 7.6 Hz, 1H), 8.07 (s, 1H ) 7.747.72 (d, J = 8.8 Hz, 1H), 7.64 (s, 1H), 7.40 (s, 2H), 7.25-7.23 (dd, J = 8.8 Hz, 1H), 6.816.79 (d, J = 8.8 Hz, 1H), 6.57 (s, 2H), 6.41 (s, 1H), 6.17-6.13 (t, J = 8.8 Hz, 1H), 5.35 (s, 1H), 4.76 (s, 2H), 4.41 (m, 1H), 4.18-4.16 (t, J = 8 Hz, 1H), 3.87-3.85 (tt, 1H), 3.79-


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3.76 (d, J = 12 Hz,1H), 3.64-3.62 (m, J = 10.4 Hz, 1H), 3.18(s, 3H), 2.22-2.20 (t, J = 6 Hz, 2H), 1.94-1.93 (t, J = 6.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 176.87, 174.29, 166.70, 163.39, 163.22, 163.11, 155.52, 154.65, 151.90, 151.38, 149.58, 146.48, 145.06, 129.35, 129.62, 121.83, 121.39, 111.55, 96.41, 84.87-83.72, 81.56, 68.78-68.34, 59.10, 55.80, 55.23, 40.79, 34.04, 27.14. ESI-MS m/z calcd [M-H+]: 699.2325. Found: 698.2197. Formation of MTX-GEM ASMP-NPs. At room temperature, 5 mg MTX-GEM ASMP was dissolved in 2 mL DMF. Then the solution was stirred for 0.5 h. Subsequently, the DMF solution of MTX-GEM ASMP was added dropwise into the 4 mL deionized water and then the resulting solution was stirred vigorously for another 0.5 h. In order to remove any residual DMF, the solution was dialyzed against deionized water for 24 h (molecular weight cutoff = 3500 g mol-1), and the water was renewed every 4 h. After the process of dialysis, the obvious opalescence appeared in water, which confirmed the formation of MTX-GEM ASMP-NPs. Measurement of CAC. The CAC value of MTX-GEM ASMP-NPs was investigated by the fluorescence probe technique using NR as the fluorescence probe.48 Typically, the various concentrations of MTX-GEM ASMP-NPs in deionized water (from 22.0 nM to 100.0 μM) were prepared. 25 μL of NR acetone solution (1.6 × 10-4 M) was injected into 4 mL of MTX-GEM ASMP-NP solution at various concentrations. Afterward, the resulting solution was placed overnight to evaporate and remove the acetone. The final concentration of NR was fixed at 1.0 × 10-6 M in each sample. Ultimately, the fluorescence emissions of NR were measured with the exciting



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wavelength of 550 nm. In Vitro Drug Release Study. In vitro release behavior of MTX-GEM ASMP-NPs was evaluated under simulated physiological conditions by dialysis in PBS (pH 7.4) containing (or not) 25% FBS and in PBS (pH 5.0) at 37 °C containing (or not) 25% FBS respectively, which were chosen to mimic the extracellular and lysosomal compartments. The rate of GEM release was used as a predictor to determine the hydrolytic stability of the conjugate in simulated media.17,49,50 MTX-GEM ASMP-NPs (0.5 mg mL-1) were prepared as described above. Typically, a total of 3 mL MTX-GEM ASMP-NPs solution was transferred into a dialysis bag (MWCO = 500 g mol-1). Afterward, the dialysis bag was immersed in 60 mL PBS (pH 7.4) containing (or not) 25% FBS or 60 mL PBS (pH 5.0) containing (or not) 25% FBS with gentle agitation at 37 °C in the dark. At specific time intervals, 1 mL of the external buffer was withdrawn and replaced with 1 mL fresh media immediately. Here, the external buffer was analyzed by HPLC using an XBridge C18 reverse phase column (4.6, 150 mm, 5 μm) with UV detection at 268 nm at 25 °C. Acetonitrile-water (3%-97%) was applied and the flow rate was set at 0.8 mL min-1. During the assay, 20 μL of each sample was injected into the analytic column. Preparation of NR-Loaded MTX-GEM ASMP-NPs. In a typical procedure for the preparation of NR-loaded MTX-GEM ASMP-NPs: 2 mg MTX-GEM ASMP was dissolved in 1 mL of DMSO, followed by adding 0.4 mL of NR solution (0.1 mg mL-1) in DMSO and stirring at room temperature for 1 h. Then the mixture was slowly added into 4 mL of deionized water and stirred slightly for another 1 h. Subsequently, the


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solution was dialyzed against deionized water for 48 h (molecular weight cutoff = 1000 g mol-1) and the deionized water was renewed every 4 h. Cell Cultures. CAL-27 cancer cells and MCF-7 cells were incubated in DMEM under a humidified atmosphere containing 5% CO2 at 37 °C. The culture medium consisted of penicillin, 10% FBS, and streptomycin. In Vitro Degradation Experiment of MTX-GEM ASMP-NPs. The CAL-27 cells at a cell density of 5.0 × 105 were cultured in 6-well plates for 24h at 37 °C. After that, were with MTX-GEM ASMP-NPs at a concentration of 35 M treated the cells for 24 h at 37 °C. Subsequently, the cells were rinsed with cold PBS and mechanically scraped by cell scrapers. The cell suspensions were sonicated for 5 min by ultrasonic cell disrupter (Vibra cell 750) and centrifuged at 4 °C. Then the supernatant was acquired and freeze-dried. The obtained sample was dissolved in methyl alcohol and analyzed by LC-MS. Cellular Uptake of MTX-GEM ASMP-NPs in CAL-27 cells. Flow cytometry and CLSM were applied to perform the experiments for cellular uptake of MTX-GEM ASMP-NPs in CAL-27 cells. For flow cytometry, CAL-27 cells were seeded in the 6well dishes at a density of 5.0 × 105 cells well-1 and incubated for 24 h. The solutions of NR-loaded MTX-GEM ASMP-NPs （35 µM） were prepared. Thereafter, the cells were incubated in 2 mL of fresh medium containing NR-loaded MTX-GEM ASMPNPs (35 µM) for the predetermined time intervals (15 min, 0.5 h, 1 h, 2 h and 4 h) in CO2 incubator. After the removal of culture media, the cells were detached from the wells by trypsinization for flow cytometry analysis. Data for 1.0 × 104 gated events



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were collected and analysis was performed for cell internalization efficiency by means of a BD FACSCalibur flow cytometer. For the CLSM studies, CAL-27 cells were seeded on the slides placed in the 6-well dishes (5.0 × 105 cells well-1) and allowed to attach for 24 h. Then, the culture medium was replaced by 2 mL fresh medium containing NR-loaded MTX-GEM ASMP-NPs (35 µM). The cells were cultured for the predetermined time intervals (15 min, 0.5 h, 1 h, 2 h and 4 h) in CO2 incubator. Subsequently, culture media was removed, and the slides were washed with cold PBS. After that, 4% paraformaldehyde were used to fix the cells for 0.5 h at 25 °C. Thereafter, the cells were permeabilized by 0.1% Triton X-100 solution on ice for 15 min. Afterward, the cells were treated by RNAse for 20 min at 37 °C. Subsequently, 1 mL of 2.4 nM YOYO-1 was added to stain cell nucleus for 15 min. Finally, the slides were mounted with antifade solution. The resulted slides were imaged by a LEICA TCS SP8 fluorescence microscopy. Cytotoxicity Measurements of MTX-GEM ASMP-NPs. MTT viability assay was utilized to estimate the anticancer activities of MTX-GEM ASMP-NPs against MCF-7 cells, MCF-7/ADR cells and CAL-27 cells. MTX, GEM and the MTX+GEM mixture (1:1) were taken as controls. The CAL-27 cells at a cell density of 7 ×103 per well were seeded into 96-well plates and incubated for 24 h at 37 °C. After that, the culture medium was replaced by 200 µL of new medium containing different drug formulations (MTX-GEM ASMP-NPs, MTX, GEM, or MTX+GEM mixture). The cells were incubated for additional 72 h. Thereafter, 20 μL of MTT assay stock solution (5 mg mL1

) was added into each well and incubated for an additional 4 h to form formazan.


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Subsequently, the medium containing unreacted MTT in each well was removed carefully and 200 μL DMSO was added to dissolve the obtained blue formazan crystals. Finally, a BioTek Synergy H4 hybrid reader was utilized to measure the absorbance of the resulting solution at 490 nm for the cell viability. Determination of Synergistic Effect of MTX-GEM ASMP-NPs. The CI method of Chou and Talalay51 was used to analyze the nature of the interaction between MTX and GEM at various levels of cytotoxicity using Calcusyn software. CI, grounded on the median-effect equation and derived from the dose effect profiles of a given drug combination, is defined for mutually nonexclusive treatment regimens as: CI = D1/Dx1 + D2/Dx2 where D1 and D2 represent the doses of drug 1 and drug 2 alone, that of the two combined required to achieve the particular fa (e.g., 50% inhibition of cell viability). Dx1 and Dx2 represent the doses for single drugs which are required to achieve a same fa. Levels of interaction are defined as follows: CI greater than 1.3 indicates antagonism, CI between 1.1 and 1.3 indicates moderate antagonism, CI between 0.9 and 1.1 indicates additivity, CI between 0.8 and 0.9 indicates slight synergy, CI between 0.6 and 0.8 indicates moderate synergy, CI between 0.4 and 0.6 indicates synergy, and CI less than 0.4 indicates strong synergy.24 Generally, CI values are plotted against drug effect levels (ICx values), from which quantitative information about the nature and extent of drug interactions can be demonstrated. The concept of DRI was formally put forward by Chou JH and Chou TC in 1988.25 The DRI is a measure of how many folds the dose of each drug in a synergistic



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combination may be reduced at a given effect level, comparing with the doses of each drug alone.23 Thus, for two-drug combinations CI = D1/Dx1 + D2/ Dx2 = 1/DRI1 + 1/DRI2, the DRI is of great importance in clinical manifestations, in which dose reduction leads to reduced toxicity toward the host while the therapeutic efficacy is retained.23 Activity Assay of Caspase-3 Protein. CAL-27 cells at a density of 2.5 × 106 were seeded in culture dishes and allowed to attach for 24 h. MTX, GEM, MTX+GEM mixture and MTX-GEM ASMP-NPs (35 µM) treated the CAL-27 cells for 48 h, separately. The cells without treatment were taken as a control. After the treatment, cells were rinsed with cold PBS, and collected by a cell scraper. Subsequently, 200 L of cell lysis buffer were used to disperse the cells and keeping them at 0 °C for 0.5 h. Thereafter, the samples were centrifuged at 10000 rpm for 100 s at 4 °C to obtain the lysates. The content of protein in the lysates were determined by BCA protein assay and then adjusted to the same concentration with the lysis buffer. Equal volumes of the protein solution were allocated into the wells of a 96-well plate respectively. 5 L of the DEVD-p NA caspase-3 substrate and 50 L of 2 × reaction buffer (containing 10 mM DTT) were added into each sample. Subsequently, the samples were incubated at 37 °C for 2 h and then the absorbance at 405 nm of each sample was measured with a BioTek Synergy H4 hybrid reader. The activity of caspase-3 protein was expressed as fold of the measured optical density (OD) obtained from the untreated control cells, and all the values of samples should subtract the OD value of the blank. Cell Apoptosis and Cell Cycle Assay. Cell Apoptosis and cell cycle alterations induced by treatment of MTX, GEM, MTX+GEM mixture or MTX-GEM ASMP-NPs


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were studied by flow cytometry analysis. CAL-27 cells (5.0 × 105 cells per well) were incubated in 6-well plates overnight. Then, MTX, GEM, MTX+GEM mixture or MTXGEM ASMP-NPs at the same concentration (35 µM) treated the cells for 48 h. The untreated CAL-27 cells were utilized as a control. To quantify the ratio of apoptosis, treated cells were obtained by trypsinization, rinsed twice with ice-cold PBS and resuspended in 1X binding buffer. After that, according to the manufacturer’s instructions, the cells were stained with Alexa Fluor® 488 annexin V and PI. For the determination of cell cycle alterations, the cells were collected by trypsinization and centrifugation (1500 rpm, 5 min). Then, the pelleted cells were washed twice with icecold PBS, fixed with 70% ethanol at 4 °C overnight and treated with Rnase A for 0.5 h at 37 °C. Subsequently, the hypotonic propidium iodide (PI) solution (500 g mL-1) treated the fixed cells for 0.5 h at room temperature to stain the DNA. Flow cytometry (BD FACSCalibur, USA) was applied to analyze the cell cycle status. Extraction and Detection of Intracellular dCTP, dTTP and dATP. CAL-27 cells were plated in 3.5 cm dishes at a density of 3.0 × 106 and cultured to adhere. MTX, GEM, MTX+GEM mixture or MTX-GEM ASMP-NPs treated the CAL-27 cells at the same concentration (35 µM) for 48 h, respectively. After that, the medium was removed and HEPES (1 M) was used to wash the cells. The adherent cells were detached by trypsin and suspended gently in 5 mL of ice-cold HEPES. A 20 μL aliquot was used to determine cell number via cell counting chamber. Thereafter, the samples were centrifuged for 10 min at 3000 rpm at 4 °C following by discarding the supernatants. And then the cell pellets were resuspended in 1 mL of ice-cold 60% methanol, vortexed



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vigorously and sonicated for 5 min using ultrasonic cell disrupter. The extracts were centrifuged (15000 rpm for 15 min at 4 °C) to remove cell debris, precipitated protein and DNA. The filtrate was evaporated under centrifugal vacuum at -70 °C and the resultant pellet was resuspended in 100 μL methanol : water (1:1) and analyzed by Ultra Performance

Liquid

Chromatography

&

Quadrupole-Time-of-Flight

Mass

Spectrometer (ACQUITYTM UPLC & Q-TOF MS Premier). 300 ng mL-1 of 5F-dUTP was served as internal quality control standards.

ASSOCIATED CONTENT Supporting Information

Materials; measurements; UV-Vis and fluorescence characterization data of the MTXGEM ASMP; in vitro cell degradation. This information is available free of charge on the ACS Publications website at http://pubs.acs.org/. AUTHOR INFORMATION Corresponding Author * Fax: +86-21-54741297.

E-mail: [email protected]; [email protected].

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS. This work was financially supported by the National Basic Research Program of China (2015CB931801), and National Key Research and Development Plan of China


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Self-Delivery Nanoparticles of Amphiphilic Methotrexate-Gemcitabine Prodrug for Synergistic Combination Chemotherapy via Effect of Deoxyribonucleotide Pools Yao Wang, Ping Huang, Minxi Hu, Wei Huang, Xinyuan Zhu,* and Deyue Yan*


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