Synergistic Combination Chemotherapy of Camptothecin and

Oct 26, 2015 - LC-MS was performed using a Waters Acquity UPLC system equipped with a solvent manager, a sample manager, and a high-resolution mass sp...
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Synergistic Combination Chemotherapy of Camptothecin and Floxuridine through Self-Assembly of Amphiphilic Drug−Drug Conjugate Minxi Hu, Ping Huang, Yao Wang, Yue Su, Linzhu Zhou, Xinyuan Zhu,* and Deyue Yan* School of Chemistry and Chemical Engineering, Shanghai Key Lab of Electrical Insulation and Thermal Aging, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China S Supporting Information *

ABSTRACT: Combination chemotherapy has been widely applied in cancer treatment; however, the cocktail administration of combination chemotherapy could cause the nonuniform biodistribution of anticancer agents, thus impairing the therapeutic efficacy. In the present study, to address this concern, we proposed a novel strategy of preparing self-assembled nanoparticles from amphiphilic drug−drug conjugate for synergistic combination chemotherapy. The conjugate was synthesized by two-step esterification of hydrophobic camptothecin (CPT) and hydrophilic floxuridine (FUDR) through a linker compound. Because of its amphiphilic nature, the CPT-FUDR conjugate self-assembled into stable nanoparticles which could simultaneously release fixed dosage of the two drugs in cancer cells. In vitro studies demonstrated synergistic anticancer efficacy of the CPT-FUDR nanoparticles including improved cell apoptosis, varied cell cycle arrest, as well as effective inhibition of cancer cell proliferation.

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INTRODUCTION Since the 1960s, combination chemotherapy has been intensively researched and regarded as an alternative strategy for overcoming the limitations of monotherapy including drugmitigating response, serious side effects, and poor therapeutic efficacy.1,2 The rationale for combination chemotherapy is to use drugs with different mechanisms of treatment and to deliver multiple drugs at their maximum tolerated doses.3 It is increasingly recognized that through the proper drug combination the treatment can achieve synergistic cytotoxicity in vitro and in vivo.4,5 However, due to the present cocktail administration of combination chemotherapy, the unique pharmacokinetics of individual drugs, which could give rise to noncoordinated biodistribution and suboptimal drug combination at the tumor sites,6,7 have limited the enhancement of therapeutic efficacy. Consequently, new strategies are emerging for precise and controlled delivery of multiple therapeutic agents that brings satisfactory clinical results. Rapid advances in nanodelivery systems including liposomes, nanogels, polymeric nanoparticles, peptide−drug conjugates, and inorganic mesoporous materials have opened up unparalleled opportunities in simultaneously delivering anticancer agents with distinct pharmacokinetic mechanisms via enhanced permeability and retention (EPR) effect.8−16 With the help of different carries, various nanostructures containing anticancer drugs can be obtained.17−19 Instead of using drug carriers, very recently we proposed a novel carrier-free drug delivery system based on the self-assembled nanoparticles from amphiphilic drug−drug conjugate (ADDC), in which the anticancer drugs can be delivered into the sites of action by themselves. Once drug− © XXXX American Chemical Society

drug conjugate is biodegraded in the tissues or cells, free anticancer drugs are released to work on the diseased regions simultaneously, resulting in high anticancer efficacy.20 In the present study, the ADDC concept is further extended into synergistic combination chemotherapy. Both hydrophilic and hydrophobic anticancer drugs with synergistic effect are conjugated onto a linker to form the drug−drug conjugate. Because of its amphiphilicity, the conjugate can self-assemble into nanoparticles to offer pharmacodynamic and pharmacokinetic advantages, realizing the combination chemotherapy of two anticancer drugs with different physicochemical properties. To clarify our idea, we chose hydrophobic camptothecin (CPT) and hydrophilic floxuridine (FUDR) as the chemotherapeutic agents for synergistic combination chemotherapy. CPT is a hydrophobic, topoisomerase-1 inhibitor first isolated from the Chinese tree Camptotheca acuminate in the 1960s, it exhibits a broad range of anticancer activity via the function of binding to the topoisomerase-1 and DNA complex.21,22 Unfortunately, low aqueous solubility and unpredictable efficacy of CPT limit its clinical use.23,24 FUDR, a derivative of 5-fluorouracil, is known for its high antitumor activity against cancer metastases.25,26 Besides, the high solubility of FUDR in water could compensate the extreme hydrophobicity of CPT. Scientists have discovered that FUDR and irinotecan, which is a semisynthetic prodrug of CPT, exhibit remarkable synergistic anticancer effect in gastrointestinal tumor lines at the ratio of Received: September 22, 2015 Revised: October 24, 2015

A

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Figure 1. Synthetic route of CPT-FUDR conjugate and its self-assembly into nanoparticles for combination chemotherapy.

1:1.27,28 Therefore, we hypothesized that CPT and FUDR could provide synergy in a human gastrointestinal cancer cell line. Here, CPT and FUDR were connected to the diglycolic anhydride linker through esterification, and the resultant amphiphilic conjugate could self-assemble into nanoparticles. Once the nanoparticles entered the cancer cells, they would release a precise ratio of two anticancer drugs by simple hydrolysis. This characteristic of our combination chemotherapeutic strategy is strongly critical because the drug ratio in the cancer cells can govern whether the combination is synergistic or antagonistic. The therapeutic effect on human colon cancer cells was evaluated in vitro, and the results demonstrated that the self-assembled CPT-FUDR nanoparticles held the potential to be further engineered as a combinatorial drug delivery system with synergistic therapeutic function.

in CPT-FUDR) and 4.60 (11 in CPT-FUDR) in the 1H NMR spectrum of the CPT-FUDR conjugate are assigned to methylene protons of OOCCH2OCH2COO, respectively. For the 13C NMR in Figure 2B, the peak at 72.84 ppm (18 in CPT) corresponding to HO−CCO− (lactonic ring) of CPT shifts to 77.15 ppm (18 in CPT-FUDR) in the 13C NMR spectrum of the CPT-FUDR conjugate and the peak at 70.58 ppm (9 in FUDR) corresponding to −CH2−OH of FUDR shifts to 64.93 ppm (25 in CPT-FUDR). In addition, the carbon signals appear at 169.52 ppm (21 in CPT-FUDR) and 167.71 ppm (24 in CPT-FUDR) attributed to −OCO− in the 13C NMR spectrum of the CPT-FUDR conjugate further confirm the esterification. Liquid chromatography−mass spectrometry (LCMS) was used to determine the exact mass and purity of the CPT-FUDR conjugate, and the result is shown in Figure 3. According to the liquid chromatogram, CPT-FUDR conjugate has a retention time of 3.91 min with high purity. The MS analysis shows that the m/z ratio of CPT-FUDR is 693.1832, which is in accordance with the calculated m/z ratio of 693.1766. These results underline the successful synthesis of CPT-FUDR conjugate. Other characterizations of CPT-FUDR conjugate including Fourier transform infrared (FTIR) spectroscopy, ultraviolet−visible (UV−vis) absorption spectroscopy, and fluorescence spectroscopy were also conducted as shown in Figures S2 and S3. Through FTIR analysis, the hydroxyl stretching vibration of CPT at about 3433 cm−1 and that of FUDR at 3312 cm−1 disappear because of the formation of ester linkage. The absorption peak at 1752 cm−1 in the spectrum of CPT-FUDR conjugate belongs to the −CO stretching absorption. Figure S3A compares the UV−vis spectra of CPT, FUDR, and CPT-FUDR conjugate. It can be found that the spectrum of CPT-FUDR conjugate contains the major UV−vis absorptions of both free drugs. Compared to the absorption of CPT, a blue shift of the absorption maximum from 363 to 360 nm can be observed from the absorption of the CPT-FUDR conjugate. Because FUDR has no fluorescence property, the fluorescence spectrum of CPT-FUDR conjugate in Figure S3B is similar to that of CPT. The maximum emission

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RESULTS AND DISCUSSION Synthesis and Characterization of CPT-FUDR. The synthetic route of CPT-FUDR is given in Figure 1. The CPTCOOH was obtained by esterification of CPT and diglycolic anhydride using 4-(dimethylamino) pyridine (DMAP) as catalyst, in which the molar feed ratio of CPT to diglycolic anhydride was 1:5 due to steric hindrance. The 1H NMR spectroscopy of CPT-COOH is shown in Figure S1. Compared with the 1H NMR spectroscopy of CPT, the peak at 6.52 ppm (11 in CPT) associated with the hydroxyl proton disappears completely and the new peak at 4.10 ppm (13 in CPT-COOH) belongs to the terminal COOH of the CPT-COOH. The reaction of CPT-COOH and FUDR was carried out using N,N′-dicyclohexylcarbodiimide (DCC) and DMAP catalyzing esterification, and the structure of the resulting CPT-FUDR conjugate was confirmed by 1H NMR and 13C NMR spectroscopy (Figure 2). For the 1H NMR in Figure 2A, the peak at 5.15 ppm (8 in FUDR) ascribed to the hydroxyl proton of the FUDR disappears obviously and the signal at 3.78 ppm (7 in FUDR) attributed to α-methylene protons of FUDR shifts to 4.20 ppm (13 in CPT-FUDR). The peaks at 4.43 (12 B

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Figure 2. (A) 1H NMR (400 MHz, DMSO-d6) spectra and (B) conjugate.

13

C NMR (100 MHz, DMSO-d6) spectra of CPT, FUDR, and CPT-FUDR

peak of CPT is 430 nm, while the maximum emission of the CPT-FUDR conjugate blue-shifts to 426 nm due to the

formation of ester linkages. All these characterizations indicate the successful synthesis of CPT-FUDR conjugate. C

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that the CPT-FUDR nanoparticles can remain stable for at least two weeks in storage. All these results demonstrate that CPTFUDR conjugate can self-assemble into nanoparticles in water. In Vitro Drug Release from the CPT-FUDR Nanoparticles. The in vitro release behavior of FUDR from CPTFUDR nanoparticles was tested by the dialysis method in PBS (pH = 7.4) and acetate buffered solutions (pH = 5.0) containing (or not) esterase (30 U/mL) at 37 °C. The cumulative release curves in Figure 5 show that the release of

Figure 3. Mass analysis of CPT-FUDR conjugate and liquid chromatogram (inset).

Preparation and Characterization of Self-Assembled CPT-FUDR Nanoparticles. The amphiphilic nature of CPTFUDR conjugate makes it possible to self-assemble into nanoparticles. The CPT-FUDR nanoparticles were prepared though the dialysis procedure, and the self-assembly behavior was confirmed by measuring the critical aggregation concentration (CAC) value. Here we use 1,6-diphenyl-1,3,5-hexatriene (DPH) as a UV probe. DPH partitions spontaneously into the hydrophobic region. With the increase of the CPT-FUDR nanoparticle concentration, the UV absorbance at 305 nm increased, which was plotted against the nanoparticle concentrations. As shown in Figure S4, the value of absorbance increases slowly as the concentration of CPT-FUDR nanoparticles is low. However, it increases dramatically when the concentration exceeds a certain value, indicating the formation of nanoparticles and encapsulation of DPH. The CAC value was obtained at the intersection of the tangents to the two linear portions of the graph. The CAC value is around 15 μM for the CPT-FUDR nanoparticles. The hydrodynamic size and size distribution of the CPT-FUDR nanoparticles were evaluated by dynamic light scattering (DLS), and the morphology was determined by transmission electron microscopy (TEM). As shown in Figure 4A, the DLS analysis revealed a monomodal size distribution peak with a z-averaged diameter of 36.4 nm as well as a calculated PDI of 0.270, suggesting that the CPT-FUDR conjugate could form aggregates in water. Figure 4B presents the spherical morphology of the CPTFUDR nanoparticles with an average size of 30.0 nm, which is consistent with the data from DLS. Additionally, the DLS measurements were carried out at a range of time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 d) to evaluate the stability of the nanoparticles. The results in Figure S5 reveal

Figure 5. In vitro drug release profile of CPT-FUDR nanoparticles under different stimulus conditions (pH = 5.0 and pH = 7.4 with or without esterase).

CPT-FUDR nanoparticles is acid- and esterase-responsive. At pH 7.4 without esterase, the release of FUDR is about 25% over a period of 70 h, indicating that CPT-FUDR nanoparticles remain stable in the physiological condition. When pH is changed to 5.0, FUDR is released more rapidly from the nanoparticles. When treated with esterase, the release rate is remarkably promoted in both pH conditions. Especially at pH 5.0 with esterase, the cumulative amount of released FUDR is up to 65%. These results are consistent with the fact that ester linkage degrades much more quickly with acid and esterase. In addition, the experiment of intracellular degradation was performed to further examine whether the CPT-FUDR nanoparticles could indeed degrade into free CPT and FUDR in cells. After 6 h treatment of CPT-FUDR nanoparticles, the cellular extracts of HT-29 cells were analyzed with the LC-MS technique. As shown in Figure S6, the mixed standard drugs were used as a control. The LC-MS analysis from Figure S7 clearly confirm the existence of free CPT, FUDR, and CPTFUDR conjugate in the extraction from HT-29 cells treated

Figure 4. (A) DLS measurements of the CPT-FUDR nanoparticles in aqueous solution. (B) TEM image of CPT-FUDR nanoparticles. D

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Figure 6. Cell internalization of CPT-FUDR nanoparticles observed by confocal laser scanning microscopy. Confocal laser scanning microscopic images of HT-29 cells treated with (A) CPT/FUDR mixture and (B) CPT-FUDR nanoparticles for 30 min, 2 h, and 4 h. Cells nuclei were stained with PI (red). Scale bar = 20 μm.

Figure 7. (A) Viability of HT-29 cells treated with CPT, FUDR, CPT/FUDR mixture, and CPT-FUDR nanoparticles at different doses. (B) Combination index (CI) of CPT and FUDR combinations via different formulations.

reason these two forms of drug formula are taken up by cells with different efficiencies is that they enter in two distinct ways. As for the CPT/FUDR mixture, the drug molecules enter into cells through diffusion. Meanwhile, the CPT-FUDR nanoparticles are internalized through endocytosis, so the drug molecules could enter into cells faster within a short time. Moreover, the intracellular uptake behavior of CPT-FUDR nanoparticles was further investigated by flow cytometry. The HT-29 cells were cultured with Nile red-loaded CPT-FUDR nanoparticles (50 μM) at 37 °C for 30 min, 2 h, and 4 h, respectively; cells grown in complete culture medium were used as control that showed the autofluorescence. Figure S8 shows the histograms of cell associated Nile red fluorescence for HT29 cells. Only a small amount of fluorescence intensity is observed in the cells after 30 min incubation; while after 2 h incubation, the apparent increase of fluorescence intensity indicates that more CPT-FUDR nanoparticles have entered into cells. Therefore, these results also confirm the effective internalization of CPT-FUDR nanoparticles by cells. In Vitro Cytotoxicity and Combination Index Analysis. The CPT-FUDR nanoparticles’ ability to attenuate the proliferation of tumor cells was evaluated by 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. HT-29 cells were incubated with a series of concentrations (0.25 to 64 μM) of CPT, FUDR, CPT/ FUDR mixture, and CPT-FUDR nanoparticles and the control was complete culture medium treatment. Figure 7A shows the

with CPT-FUDR nanoparticles for 6 h. These intracellular degradation results demonstrate that the ester bonds connecting CPT and FUDR are facilely cleaved through hydrolysis within the cancer cells and free CPT and FUDR could be thereby released from the nanoparticles. Cell Internalization. To observe the intracellular uptake behavior of CPT-FUDR nanoparticles, both confocal laser scanning microscopy and flow cytometry were performed. According to the fluorescence spectral analysis, we know that both CPT and CPT-FUDR conjugate emit blue fluorescence under UV-lamp irradiation (Figure S3B), so they could be identified by the blue fluorescence in the cell internalization study. With respect to the confocal laser scanning microscopy experiment, the HT-29 cells were treated with CPT/FUDR mixture and CPT-FUDR nanoparticles (50 μM) for 30 min, 2 h, and 4 h at 37 °C. Here, the cell nuclei were stained by propidium iodide (PI). As shown in Figure 6A, the blue fluorescence of CPT can be observed in the cells after 30 min and the intensity gradually increases with prolonging the incubation time. However, at 30 min, very little blue fluorescence of CPT-FUDR nanoparticles is observed in the cells (Figure 6B) compared to CPT/FUDR mixture, suggesting that few nanoparticle has entered into cells during that period. From 2 to 4 h, as can be noticed from the merged image, the blue fluorescence grows strong and is localized in both cytoplasm and nuclei. These results establish that CPTFUDR nanoparticles could be internalized by the cells. The E

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Figure 8. (A) Cell apoptosis analysis of HT-29 cells assessed with flow cytometry analysis; the HT-29 cells were treated with CPT, FUDR, CPT/ FUDR mixture, and CPT-FUDR nanoparticles at the same concentration (10 μM) for 48 h. In each flow cytometry plot, the lower right quadrant depicts early stage of apoptotic cells, while the upper right quadrant depicts late stage of apoptotic cells, the percentages in each quadrant represent the percent of cells in that stage. (B) Cell cycle distribution of HT-29 cells assessed with flow cytometry analysis. Cells were treated with CPT, FUDR, CPT/FUDR mixture, and CPT-FUDR nanoparticles at 10 μM for 48 h.

same dose combination of the two free drugs. These results strengthen the above explanation that, compared to the free drug combination, CPT and FUDR released from the CPTFUDR nanoparticles in cells could remain the same molar ratio of 1:1, which interacts more synergistically than the free drugs diffused into the cells. Cell Apoptosis and Cell Cycle Analysis. To determine whether the observed inhibition of cancer cell proliferation by the CPT-FUDR nanoparticles was due to apoptosis, the annexin V/PI double staining assay was performed to detect the apoptosis ratio of cells. HT-29 cells were treated with CPT, FUDR, CPT/FUDR mixture, and CPT-FUDR nanoparticles at the concentration of 10 μM. After 48 h incubation, the cells were then processed by annexin V/PI double staining. Here, cells grown in complete culture medium were used as control. In Figure 8A, the flow cytometry analysis show that after treatment of CPT, FUDR, CPT/FUDR mixture, and CPTFUDR nanoparticles, the percentages of apoptotic HT-29 cells are 15.9%, 9.6%, 18.4%, and 37.1%, respectively. These results demonstrated that CPT-FUDR nanoparticles induce a much higher level of apoptosis in HT-29 cells compared to the other formulations at the same concentration. To further explore the effect of CPT-FUDR nanoparticles on cell proliferation, we also evaluated cell cycle arrest. For cell cycle analysis, HT-29 cells were treated with CPT, FUDR, CPT/FUDR mixture, and CPT-FUDR nanoparticles at the same concentration (10 μM) for 48 h and then stained with PI to investigate their DNA content by flow cytometry. The results in Figure 8B show that the percentages of cells in G2/M phase all increase when HT-29 cells are treated with indicated drug formulations. Notably, when treated with CPT-FUDR nanoparticles, the increase in the percentage of cells at G2/M phase is very noteworthy (from 6.24% to 29.29%) along with corresponding reduction in the percentage of cells in the G0/G1 phase. In the meantime, an obvious sub-G0/G1 apoptotic phase is observed. These data clearly reveal that CPT-FUDR nanoparticles induce the cell cycle arrest in HT-29 cells at the G2/M phase and they are consistent with apoptosis results.

cell viability after 48 h incubation with four different drug formulations at various concentrations. Obviously, CPT-FUDR nanoparticles exhibit significant inhibition to the cell proliferation (IC50 = 0.15 μM) compared to that of the other drug formulations (IC50,FUDR = 95 μM, IC50,CPT = 2.3 μM, IC50,CPT/FUDR = 1.24 μM), particularly at low concentrations. Although the CPT/FUDR mixture contains the same drug dosage as the nanoparticles, it exhibits less cell toxicity. The explanation for this result could be CPT-FUDR nanoparticles being effectually internalized by the cells as well as a fixed ratio of drugs with remarkable synergistic effects being successfully released from the nanoparticles. To further study the synergy in the CPT/FUDR mixture and in CPT-FUDR nanoparticles, the combination index (CI) method that could provide a simple way to quantitate the drug interaction information was carried out. CI, based on the median-effect equation and derived from the mass-action law principle, is defined as13,29 CI =

D DA + B DmA DmB

where DA and DB indicate the doses of drug A and drug B, respectively, that in combination exert some particular effect (e.g., 30% inhibition of cell viability). DmA and DmB indicate the doses for single drugs at which the drugs can achieve the same effect. Values of CI > 1 represent antagonism, CI = 1 represent additive, and CI < 1 represent synergism. Usually, 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. Figure 7B shows the comparison of CI values between CPT/FUDR mixture and CPT-FUDR nanoparticles; most of the data points are below the line of CI = 1 with 48 h incubation of drugs; only a few data points from CPT/FUDR mixture are above or near the CI = 1 line. In addition, the CI values of CPT-FUDR nanoparticles are much lower than those of CPT/FUDR mixture at each corresponding ICx value, denoting that the synergistic effect of CPT-FUDR nanoparticles is more remarkable than just the F

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Bioconjugate Chemistry Caspase-3 Expression Regulated by CPT-FUDR Nanoparticles. Caspases, as a family of cysteine-aspartyl proteases, are well recognized for their critical roles in apoptosis and inflammation.30,31 Among them, caspase-3 is considered to be the central effector in the caspase-dependent apoptotic pathway, and it is reported as being activated in response to cytotoxic drugs.32,33 To examine the involvement of caspase-3 in CPT-FUDR nanoparticle treated cells, the expression of caspase-3 was quantified using a fluorogenic caspase-3 substrate, DEVD-pNA. HT-29 cells were treated with CPT, FUDR, CPT/FUDR mixture, and CPT-FUDR nanoparticles at the same concentration of 10 μM for 48 h, and cells grown in complete culture medium were set as a blank control. Figure 9

combination using CI method. In vitro studies exhibit that compared to CPT/FUDR mixture, CPT-FUDR nanoparticles could more effectively induce the apoptosis of HT-29 cells mediated by activation of caspase-3 and inhibit the cell growth at the G2/M phase. Given these advantages of CPT-FUDR nanoparticles in anticancer efficacy, we believe that this work could offer a new strategy for combination chemotherapy delivery of other anticancer drugs exhibiting opposite hydrophilicity and hydrophobicity with high efficiency and more profound therapeutic effect.

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EXPERIMENTAL SECTION Materials. CPT (99%) was purchased from Dalian Melone Biotechnology Ltd. FUDR (99%) and methanol (LC-MS, ≥99.9%) were purchased from Aladdin Industrial Corporation. Diglycolic anhydride (99%) was obtained from Alfa Aesar China Co., Ltd. DMAP (99%) and DCC (99%) were purchased from J&K Chemical Ltd. Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF) were dried by calcium hydride for more than 48 h and distilled before use. DPH (≥97.5%), esterase (from porcine liver, ≥50 U/mg), McCoy’s 5A medium, phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin−streptomycin liquid (100×), and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-FITC apoptosis assay kit for flow cytometry, bicinchoninic acid (BCA) protein assay, caspase-3 activity assay, and cell cycle assay were carried out using a fluorogenic assay kit from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals used for this study were provided by domestic suppliers and were analytical grade unless otherwise stated. Methods. 1H NMR and 13C NMR spectra were recorded on a Bruker HD 400 MHz spectrometer by using TMS in solvent of DMSO-d6 as internal standard. LC-MS was performed using a Waters Acquity UPLC system equipped with a solvent manager, a sample manager, and a highresolution mass spectrometer (Waters, USA). The FTIR spectra were detected between 4000 and 400 cm−1 using a Thermo Nicolet 6700 spectrophotometer, and all samples were cast on potassium bromide (KBr) pallet before the measurement. The UV−vis absorption spectra were recorded in the region 200−400 nm using a Thermo EV300 UV−vis spectrometer. The fluorescence spectra were recorded on a PerkinElmer LS 50B fluorescence spectrometer with the excitation at 360 nm and fluorescence from 365 to 650 nm. The DLS measurements were performed with a Zetasizer Nano S (Malvern Instruments, UK) equipped with a 4.0 mW He−Ne laser of wavelength 633 nm and positioned at a 90° scattering angle. The TEM imaging was carried out on a FEI Tecnai G2 spirit Biotwin microscope at an acceleration voltage of 200 kV. The samples for TEM observation were prepared by loading nanoparticle solution on copper grids covered with carbon film and drying in air. Synthesis of CPT-FUDR Conjugate. First we synthesized CPT-COOH according to a previously published paper with some modifications.24 CPT (348 mg, 1 mmol), diglycolic anhydride (580 mg, 5 mmol), and DMAP (122 mg, 1 mmol) were dissolved in 50 mL of dried DMSO. After stirring for 48 h at 75 °C in the dark, the solution was evaporated under reduced pressure. The crude product was purified by reverse phase column chromatograph eluting with methanol/deionized water (75:25). The purified intermediate (237 mg, yield: 51.1%) was obtained by rotary evaporation. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.15 (d, J = 8.5 Hz, 1H), 8.10 (d, J

Figure 9. Caspase-3 expression in HT-29 cells treated with CPT, FUDR, CPT/FUDR mixture, and CPT-FUDR nanoparticles at the concentration of 10 μM for 48 h.

shows that compared to control, treatment of CPT, FUDR, and CPT/FUDR mixture for 48 h results in a slightly increase in the expression levels of caspase-3 (1.82-fold, 1.77-fold, and 2.82fold). However, when HT-29 cells are treated with CPT-FUDR nanoparticles, an extraordinary up-regulation expression level of caspase-3 protein (3.39-fold) can be observed. Obviously, the CPT-FUDR nanoparticles are more effective in boosting the activation of caspase-3 in comparison with the other drug formulations, which corresponds well with the abovementioned significant synergistic effect of the CPT-FUDR nanoparticles.

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CONCLUSIONS In conclusion, we have demonstrated a synthetic strategy to conjugate two anticancer drugs with fixed synergistic ratio through a linker compound. In this study, the amphiphilic CPT-FUDR conjugate can self-assemble into nanoparticles with regular shape and stability in water. Unlike the free drug combination administration, the CPT-FUDR nanoparticles could maintain dose ratio for prolonged time and deliver hydrophobic CPT and hydrophilic FUDR simultaneously into cancer cells. The synergistic effect of CPT-FUDR nanoparticles has proven to be superior to that of free CPT and FUDR G

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Bioconjugate Chemistry = 7.9 Hz, 1H), 7.85 (t, J = 7.4 Hz, 1H), 7.69 (t, J = 7.1 Hz, 1H), 7.12 (s, 1H), 5.50 (s, 2H), 5.27 (s, 2H), 4.60 (d, J = 17.0 Hz, 2H), 4.43 (d, J = 17.0 Hz, 2H), 4.10 (s, 1H), 2.21−2.05 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H). To synthesize CPT-FUDR conjugate, CPT-COOH (232 mg, 0.5 mmol) was dissolved in a mixture of 200 mM DCC (103 mg, 0.5 mmol) and 20 mM DMAP (6 mg, 0.05 mmol) prepared in 5 mL of dried DMF. After stirring for 30 min at 0 °C, the resulting mixture was added dropwise into a 50 mL flask containing FUDR (246 mg, 1 mmol) and 10 mL DMF. Then the solution was stirred for 24 h at 40 °C under nitrogen. After that, the reaction mixture was filtered to remove dicyclohexylurea (DCU) and DMF was removed by vacuum. The residue was dissolved in 20 mL ethyl acetate, and then saturated sodium chloride solution was used to wash the ethyl acetate solution three times. After that the organic layer was collected, dried over anhydrous magnesium sulfate, and evaporated to afford the crude product, which was purified by column chromatography eluting with ethyl acetate/ methanol (95:5) to give CPT-FUDR conjugate (276 mg, yield: 80.0%) as an orange solid. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.81 (d, J = 4.9 Hz, 1H), 8.66 (d, J = 12.4 Hz, 1H), 8.12 (m, J = 16.9, 8.5 Hz, 2H), 7.85 (t, J = 7.4 Hz, 2H), 7.77− 7.59 (m, 1H), 7.12 (s, 1H), 6.10 (t, J = 6.3 Hz, 1H), 5.50 (s, 2H), 5.39 (d, J = 4.4 Hz, 1H), 5.27 (d, J = 15.8 Hz, 2H), 4.60 (d, J = 17.0 Hz, 2H), 4.43 (d, J = 17.0 Hz, 2H), 4.30−4.23 (m, 1H), 4.23−4.12 (m, 2H), 3.94−3.84 (m, 1H), 2.22−2.00 (m, 4H), 1.03−0.24 (m, 3H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 169.90, 169.52, 167.71, 157.19, 153.00, 149.62, 148.55, 146.77, 145.75, 141.89−139.59 (d, J = 231 Hz), 132.23, 131.06, 130.45, 129.58, 129.18, 128.36, 119.48, 95.61, 85.17, 84.33, 77.15, 70.74, 68.17, 67.90, 67.00, 64.93, 50.91, 30.86, 8.20. ESIMS (M+H+), methanol: m/z calcd: 693.1766, observed: 693.1832. FTIR (KBr): 3443, 2934, 1752, 1704, 1662, 1617, 1564, 1502, 1455, 1403, 1384, 1352, 1264, 1234, 1204, 1138, 1087, 1052, 998, 787, 765, 723 cm−1. Preparation of CPT-FUDR Nanoparticles. At room temperature, a tetrahydrofuran (THF) solution containing the CPT-FUDR conjugate (1 mL, 1 mg/mL) was added dropwise to deionized water while stirring. Subsequently, the solution was loaded into a 1 kDa MWCO dialysis bag and dialyzed against deionized water for at least 24 h, and the water was renewed every 4 h. Determination of CAC Value. The CAC of CPT-FUDR conjugate was investigated by UV measurements using the rodshaped DPH as UV probe.34 The DPH solution in acetone (6 × 10−5 M) was mixed with CPT-FUDR nanoparticle suspensions, which ranged in concentration from 0.625 to 160 μM, and the final concentration of the DPH in each sample was 6.0 × 10−7 M. The solutions were left in the dark to equilibrate for at least 3 h before the spectroscopic measurements. The absorption spectra of the samples were recorded at 310 nm using a Thermo EV300 UV−vis spectrometer. In Vitro Drug Release Studies. Dialysis was used to investigate the release profile of FUDR from CPT-FUDR nanoparticles. The in vitro release experiments were performed at 2 different pH values (pH = 7.4 in PBS and pH = 5.0 in acetate buffered solution) with or without esterase. For each study, CPT-FUDR nanoparticles (1.5 mg) were dispersed into 3 mL of buffer solution, and the solution was loaded into a 1 kDa MWCO dialysis bag. The dialysis bags were then immersed in 100 mL of buffer solutions at 37 °C with gentle agitation. At indicated time points, concentrations of FUDR in the buffer solution surrounding the dialysis bag were

determined via UV absorbance measurement with reference to FUDR’s standard curve. External buffer solution was replenished at each time point. Esterase (30 U/mL) was added into the dialysis bag, and then drug release was studied in the presence of esterase. Cell Culture. The human colorectal cell line HT-29 was obtained from American Type Culture Collection (Rockville, MD, USA). Cells were grown in McCoy’s 5A medium supplemented with 10% FBS, 1× penicillin/streptomycin, and incubated in a humidified atmosphere containing 5% CO2 at 37 °C. Intracellular Degradation Study of CPT-FUDR Nanoparticles. The HT-29 cells were placed in 6-well plates (5.0 × 105 cells/well) with 2 mL complete McCoy’s 5A medium and grown overnight at 37 °C, then the culture medium was replaced with fresh medium that contained 50 μM of CPTFUDR nanoparticles. After 6 h incubation, cells were washed with cold PBS and harvested by trypsinization process into 15 mL polypropylene centrifuge tubes. After centrifugation at 1000 rpm for 5 min at room temperature, cells were treated with methanol. Subsequently the cell suspensions in an ice bath were disrupted by sonication with an ultrasonic cell disrupter (Vibra cell 750) for 20 min with a 40% duty cycle. Finally, the lysed cells were centrifuged at 10 000 rpm for 10 min at 4 °C and the supernatant liquid was analyzed using LC-MS. Intracellular Uptake of CPT-FUDR Nanoparticles. The ability of CPT-FUDR nanoparticles to enter cancer cells was studied with the help of confocal laser scanning microscopy and flow cytometry. For confocal laser scanning microscopy, the HT-29 cells were cultured in the 6-well dishes (5.0 × 105 cells/ well) with 2 mL complete McCoy’s 5A medium. After incubation for 24 h, the culture media were replaced with 2 mL of fresh medium containing CPT-FUDR nanoparticles and CPT/FUDR mixture (50 μM). Afterward the cells were cultivated with prearranged time (0.5, 2, 4 h) at 37 °C. For the CPT/FUDR mixture solution preparation, a final concentration of 0.5% (v/v) DMSO was added in the media; at this concentration, the DMSO had no effect on the experiment. Following the removal of culture media, cells were washed with cold PBS and then fixed with 4% formaldehyde for 30 min at room temperature. Subsequently, the cells were rinsed with cold PBS and gently permeabilized with 0.1% Triton X-100 solution. Thereafter, RNase (0.1 mg/mL) was added to each well and the cells were incubated at 37 °C for 20 min and stained with propidium iodide (PI, 1 μg/mL) for 15 min to visualize nucleus. Finally the slides were rinsed with cold PBS and sterilized water and then mounted with antifade solution. The resulted slides were imaged with a LEICA TCS SP8 fluorescence microscopy. As for flow cytometry, Nile red was used as a hydrophobic fluorescence probe. Nile red was added in the THF solution containing CPT-FUDR conjugate, followed by the formation of CPT-FUDR nanoparticles. The HT-29 cells were plated in the 6-well dishes (5.0 × 105 cells/ well) with 2 mL complete McCoy’s 5A medium and allowed to adhere for 24 h; culture medium was subsequently removed, and fresh medium containing CPT-FUDR nanoparticles (50 μM) was added. Cells were exposed to drugs for 0.5, 2, 4 h, then washed with cold PBS and detached via trypsinization. After the preparation for flow cytometry analysis, data were acquired by using a BD LSRFortessa flow cytometer. A total of 1.0 × 104 gated events per sample were recorded. In Vitro Anticancer Activity of CPT-FUDR Nanoparticles. The cytotoxicity of CPT-FUDR nanoparticles H

DOI: 10.1021/acs.bioconjchem.5b00513 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Notes

against HT-29 cells were evaluated by MTT assay. HT-29 cells were seeded on 96-well dishes at a density of 8 × 103 cells per well. The cells were allowed to attach for overnight. The culture media were then replaced by various drug formulations of CPT, FUDR, CPT/FUDR mixture, and CPT-FUDR nanoparticles. Cells were incubated for a further 48 h and then mixed with 20 μL of MTT solution (5 mg/mL). Following incubation for 4 h at 37 °C in the dark, the formazan crystals were then dissolved by the addition of 200 μL DMSO each well. The absorbance in each well was measured with a BIO-RAD Model 680 plate reader at 490 nm, and the cell viability was expressed as a percentage of the control. Cell Apoptosis and Cell Cycle Assay. The HT-29 cells were cultured in the 6-well dishes (5.0 × 105 cells/well) with 2 mL complete McCoy’s 5A medium. After 24 h incubation, the cells were incubated with the same concentration of CPT, FUDR, CPT/FUDR mixture, and CPT-FUDR nanoparticles (10 μM) for 48 h. Here, untreated HT-29 cells were used as control. After that the cells were trypsinized, rinsed with cold PBS, and resuspended in 1× binding buffer. Then, the cells were labeled with Alexa Fluor FITC-conjugated annexin V and PI with the help of manufacturer’s protocol. For cell cycle determination, cells under the same treatment of drug formulations were harvested and washed with cold PBS, and then the cells were processed with CycleTEST PLUS DNA reagent kit according to the manufacturer’s instructions. Both cell apoptosis and cell cycle were measured by a BD LSRFortessa flow cytometer, and a total of 1.0 × 104 gated events per sample were recorded. Caspase 3 Activity Assay. HT-29 cells were seeded in 3.5 cm dishes (3.0 × 106) for 24 h with 10 mL complete McCoy’s 5A medium and then treated with CPT, FUDR, CPT/FUDR mixture, and CPT-FUDR nanoparticles at the same concentration (10 μM) for 48 h. HT-29 cells grown in complete culture medium were used as control. After the removal of culture media, cells were washed with cold PBS and harvested in lysis buffer and kept on ice for 30 min. Thereafter the samples were centrifuged for 20 min at 13 000 rpm. The protein concentration of the supernatant was determined by BCA assay and adjusted to the same concentration with the lysis buffer. An equal volume of protein solution was distributed in a 96-well plate, and then the detection buffer and DEVDpNA (Asp-Glu-Val-Asp p-nitroanilide) were placed into each well. The yellow color produced by pNA from the cleaved substrate was monitored at 405 nm by a BIO-RAD Model 680 plate reader in which data points were blankly subtracted.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Xinyuan Zhu received funding from National Basic Research Program (2015CB931801) and National Natural Science Foundation of China (51473093).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00513. 1 H NMR spectra of CPT-COOH; FTIR, UV−vis, and fluorescence spectra for the CPT-FUDR conjugate; stability of CPT-FUDR nanoparticles; in vitro cell degradation and internalization (PDF)

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REFERENCES

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DOI: 10.1021/acs.bioconjchem.5b00513 Bioconjugate Chem. XXXX, XXX, XXX−XXX