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Enhanced cellular uptake and intracellular drug controlled release of VESylated gemcitabine prodrug nanocapsules
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Enhanced cellular uptake and intracellular drug controlled release of VESylated gemcitabine prodrug nanocapsules Yanfen Fang a,1 , Fang Du a,b,1 , Yanyun Xu a , Haijing Meng a,c , Jin Huang a,d , Xiongwen Zhang a , Wei Lu a , Shiyuan Liu e , Jiahui Yu a,∗ a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, College of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, PR China b Institute of Chemistry and Biochemistry, Free University Berlin, Takustr 3, 14195 Berlin, Germany c Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw 01-224, Poland d College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, PR China e Department of Diagnostic Imaging, ChangZheng Hospital, Shanghai 200003, PR China
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Article history: Received 3 November 2014 Received in revised form 15 January 2015 Accepted 13 February 2015 Available online xxx Keywords: VESylated gemcitabine Nanocapsules Intracellular drug controlled release Enhanced cellular uptake
a b s t r a c t Gemcitabine, 2 ,2 -difluoro-2 -deoxycytidine (dFdC), is the first-line antitumor agent in the treatment of pancreatic tumors. However, it possesses certain drawbacks, such as poor biological half-life resulted from rapid metabolism and the induction of resistance, leading to its restricted therapeutic potential. With the purpose of overcoming the above drawbacks, we developed a novel VESylated gemcitabine (VES-dFdC) prodrug by coupling the N4 -amino group of the pyrimidine ring of dFdC to the carboxylic group of vitamin E succinate (VES). The resulting amphiphilic compound could protect the N4 -amino group of the pyrimidine ring of dFdC from being degraded by cytidine deaminase. What is more, the prodrug was able to form nanocapsules in aqueous media (similar to the structure of cytomembrane), confirmed by transmission electron microscope (TEM). Their average particle size is about 107 nm with zeta potential of −33.4 mV measured by dynamic light scattering (DLS). VES-dFdC nanocapsules showed accelerated accumulative drug release profile in simulated lysosome environment (sodium acetate buffer pH 5 + cathepsin B, an enzyme in lysosome), due to the easily hydrolyzed property of amide bond by cathepsin B, while rather stable in PBS (pH 7.4) or sodium acetate buffer (pH 5.0) without cathepsin B, indicating their enhanced intracellular drug controlled release manner. Besides, VES-dFdC prodrug nanocapsules showed enhanced cellular uptake ability, and the amount of cellular uptake of the nanocapsules by the pancreatic cancer cell line BxPC-3 is seventy times higher than that of native gemcitabine in the first 1.5 h. Compared with free gemcitabine, VES-dFdC nanocapsules showed essentially increased growth inhibition activity against BxPC-3 cells, indicating its great potential as prodrug for pancreatic tumor therapy with improved antitumor activity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Gemcitabine, 2 ,2 -difluoro-2 -deoxycytidine (dFdC), is a pyrimidine nucleoside analog anticancer agent. It is the first-line treatment against pancreatic cancer, and often used in combination therapy for non-small lung, ovarian and breast tumors [1–3]. However, dFdC is easily metabolized intracellularly and extracellularly by cytidine deaminase (CDA) into the chemotherapeutically inactive uracil derivative, leading to a very short plasma half-life
∗ Corresponding author. Tel.: +86 21 6223 7026; fax: +86 21 6223 7026. E-mail address: [email protected] (J. Yu). 1 These authors contributed equally to this work.
[4,5]. Since dFdC is a hydrophilic compound, it requires membrane proteins, such as human equilabrative nucleoside transporter-1 (hENT1) to transport it into the cells [6]. Therefore, tumor cells that have decreased expression of nucleoside transporters are resistant to dFdC [7]. Moreover, the resistance to dFdC is the major issue in its clinical application for the treatment of pancreatic cancer [8,9]. Great efforts have been made to overcome dFdC resistance. Schirmer et al. synthesized the amino acid ester prodrugs of dFdC, which were not as sensitive as dFdC to deamination by CDA [10]. To enhance the cellular uptake of dFdC, amphiphilic prodrugs of dFdC have been constructed to circumvent this specific resistance pathway since these compounds are expected to passively diffuse across cell membranes. The uptake of dFdC-cardiolipin conjugate was proven transporter-independent, which was consistent with
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its passive diffusion through the plasma membrane [11]. Recently, squalenoylated dFdC prodrugs were synthesized, and formulated into nanoparticles. These nanoparticles were more cytotoxic than gemcitabine in two dFdC resistance cell lines [12]. Similarly, Cui et al. discovered that the 4-(N)-stearoyl dFdC solid lipid nanoparticles were more cytotoxic than dFdC in dFdC resistance related to ribonucleotide reductase M1 over-expression [13]. Vitamin E is one of the lipophilic vitamins. Vitamin E succinate (VES) possesses good antitumor activity against a wide variety of tumors by the induction of cell apoptosis [14–17], while shows little toxicity against normal cells or tissue [18]. Herein, VES-dFdC prodrug was developed by coupling the N4 -amino group of the pyrimidine ring of dFdC to the carboxylic group of VES. The structure of the prodrug was characterized by 1 H NMR and FT IR. The VES-dFdC prodrug nanocapsules were fabricated, and their physicochemical properties, such as CAC, size and drug accumulative release were investigated. And then, cytotoxicity and cellular uptake ability were evaluated in detail. 2. Materials and methods 2.1. Materials Gemcitabine and isobutyl chloroformate were purchased from Alfa Aesar. Vitamin E succinate was bought from Aladdin Chemistry Co. Lid. Triethylamine (TEA), dimethyl formamide (DMF), and tetrahydrofuran (THF) were obtained from Sinopharm Chemical Reagent Co. Ltd. TEA was dried with 4 A˚ molecular sieve and redistilled before use. DMF and THF were dried over calcium hydride and were distilled in the presence of fresh calcium hydride just before use. 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and cathepsin B were purchased from Sigma–Aldrich. 2.2. Cell line and culture Human pancreatic cancer cell line (BxPC-3, adherent cell) were purchased from Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences. BxPC-3 cells were cultured in RPMI 1640 (Gibco BRL, Paris, France), supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT), 1% penicillin and streptomycin. BxPC-3 cells were incubated at 37 ◦ C in humidified 5% CO2 atmosphere. When the cell confluence of 90% was reached, they were routinely trypsinized and subcultured. 2.3. Synthesis of VES-dFdC The VES-dFdC prodrug was synthesized as previously described with slightly modification [19]. Briefly, triethylamine (0.17 mL, 1.2 mmol) was added to a stirred solution of vitamin E succinate (0.53 g, 1.0 mmol) in 5 mL of anhydrous THF. The mixture was cooled to −15 ◦ C, and a solution of isobutyl chloroformate (0.13 mL, 1.2 mmol) in 5 mL of anhydrous THF was added dropwise. The mixture was stirred at −15 ◦ C for 30 min and a solution of gemcitabine hydrochloride (0.300 g, 1.0 mmol) and triethylamine (0.17 mL, 1.2 mmol) in anhydrous DMF (5 mL) was added dropwise to the reaction mixture at the same temperature. The reaction mixture was stirred for 72 h at room temperature, and then concentrated in vacuo. Aqueous sodium hydrogen carbonate was added, and the mixture was extracted with ethyl acetate (3 mL × 50 mL). The combined extracts were washed with water, dried over anhydrous Na2 SO4 , and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel eluting with 1–4% methanol in dichloromethane to give pure VES-dFdC as white solid (yield: 53%). IR (neat, cm−1 ) 3500–3150, 2926, 2856, 1709, 1656, 1492, 1245; 1 H NMR (400 MHz, DMSO-d6 ) 11.15 (s, 1 H,
NHCO), 8.26 (d, 1 H, H6), 7.26 (d, 1 H, H5), 6.32 (d, 1 H, OH3), 6.18 (t, 1 H, H1), 5.29 (t, 1 H, OH5), 4.18 (m, 1 H, H3), 3.86–3.68 (m, 3 H, H4 and H5), 2.91 (m, 2 H), 2.85 (m, 2 H), 2.56 (m, 2 H), 2.00 (s, 3 H), 1.92 (s, 3 H), 1.90 (s, 3 H), 1.75 (m, 2 H), 1.02 (m, 3 H), 0.84 (s, 6 H), 0.82 (s, 6 H), 0.81 (s, 3 H); 13 C NMR (100 M, acetone-d6 ) ␦ 173.4-C25 , 171.71C22 , 164.0-C17 , 155.63-C19 , 149.98-C30 , 145.87-C15 , 141.82-C32 , 127.68-C28 , 126.14-C27 , 123.93-C31 , 123.16-C29 , 118.31-C2 , 96.99C14 , 82.49-C13 , 75.75-C39 , 70.13-C4 , 69.79-C1 , 60.21-C10 , 40.14-C40 , 38.24-C54 , 38.18-C45 , 38.11-C50 , 38.03-C52 , 33.49-C43 , 32.55-C44 , 31.97-C51 , 30.50-C38 , 30.31-C24 , 29.61-C23 , 29.32-C53 , 28.84-C49 , 28.71-C38 , 25.55-C57 , 25.15-C58 , 23.01-C42 , 21.70-C37 , 21.14-C44 , 20.13-C53 , 13.20-C35 , 12.32-C33 , 12.02-C34 . MS (ESI): m/z (%): 776 [M+]. 2.4. Nanocapsule fabrication and the critical vesicle concentration The VES-dFdC (6 mg) conjugate was dissolved in 2 mL of acetone, and stirred at room temperature for 1 h, then the acetone solution was added dropwise into 6 mL of ultrapure water under constant stirring. After then, the solution was loaded into a dialysis tube (MWCO 3500) and dialyzed against 8 L (2 L × 4) of deionized water in the dark for 16 h. The critical vesicle concentration (CVC) was determined using pyrene as a fluorescence probe. The concentration of VES-dFdC varied from 7.5 × 10−5 to 1 mg/mL with fixed pyrene concentration of 6 × 10−7 mol/L. The fluorescence spectra were measured on F-4500 fluorescence spectrophotometer (Hitchi F-4500) with the excitation wavelength of 335 nm. The I373 /I384 of fluorescence intensity ratio in the emission spectra of pyrene was analyzed for the calculation of CVC. 2.5. Release of dFdC from VES-dFdC nanocapsules The rate of release of dFdC from VES-dFdC prodrug was measured with RP-HPLC system of Agilent 1200 (Agilent Technologies Inc. Shanghai Branch) using a Zorbax Eclipse XDB-C18 column (5 m, 4.6 mm × 250 mm) at 30 ◦ C. The release of the drug from VES-dFdC was performed by dissolving the conjugate 1 mg/mL in DMSO, then 30 L of the DMSO solution was dispersed into 1 mL of phosphate buffer saline (PBS) (pH 7.4), sodium acetate buffer (pH 5.0) and also sodium acetate buffer (pH 5.0) in the presence of 10 U cathepsin B. Briefly, the samples were kept in a THZ-C isothermal shaker at 37 ◦ C and 150 rpm. At predetermined time point, 100 L of solution was withdrawn from the samples for HPLC analysis using pure methanol as the mobile phase. The flow rate of the mobile phase was 1 mL/min. The Agilent 1200 Uv/vis detector was set at 248 nm. The release percentage of dFdC was calculated from the ratio of peaks area assigned to free dFdC and VES-dFdC. Experiments were carried out up to 96 h in triplicates. 2.6. In vitro tumor cell growth inhibition activity of VES-dFdC nanocapsules In vitro tumor cell growth inhibition activity of VES-dFdC nanocapsules was determined by evaluation of the viability of pancreatic cancer cell line (BxPC-3) by MTT method. Cells were seeded in 96-well plates at an initial density of 4 × 103 cells/well in 200 L RPMI-1640 culture medium and incubated for 18–24 h to reach 80% confluence at the time of treatment. Culture medium was replaced with 100 L fresh media containing various amounts of VES-dFdC conjugate, dFdC and VES (0, 0.15, 1.5, 2.9, 5.9, 11.8, 23.7 and 47.5 M). Cells were incubated for 48 h. And then, 10 L of MTT solution (0.5 mg/mL) was added into the wells. After further incubation for 4 h in incubator, 100 L of DMSO was added to each well to replace the culture medium and dissolve the insoluble
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formazan-containing crystals. The optical density was measured at 570 nm using an automatic BIO-TEK microplate reader (Powerwave XS, USA), and the cell viability was calculated from following equation: Cell viability (%) =
ODsample ODcontrol
× 100%
(1)
Where ODsample represents an OD value from a well treated with samples and ODcontrol from a well treated with PBS (0.01 M, pH 7.4) buffer only. Values were reported as the means for each triplicate sample. Means and corresponding standard deviations (mean ± SD) were shown as results.
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Fluorescence measurement was performed on a Hitachi F-4500 Fluorescence Spectrophotometer, samples were dissolved in pure water, and pyrene was used as the fluorescent probe. The particle sizes, size distributions and zeta () potentials of nanocapsules were measured by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, UK). Morphological evaluation of the nanocapsules was obtained with a Philips CM 120 transmission electron microscope (TEM), working at 60-kV accelerating voltage. A 10 L of capsule solution of 10−1 mg/mL was carefully dropped onto clean copper grids, and then dried at room temperature for 30 min before imaging on the microscope. The average size of the capsules was determined from the TEM images and the scale bars were sharpened with Photoshop 8.0 software.
2.7. In vitro evaluation of cellular uptake 3. Results and discussion The amount of the uptake of VES-dFdC nanocapsules by the BxPC-3 cells was measured by the HPLC with free dFdC as a control in this experiment. Cells were seeded in 6-wells plate at an initial density of 2.5 × 105 cells per well in 2 mL of culture medium. After overnight incubation, the old culture medium was displaced by 2 mL of fresh medium which contains VES-dFdC nanocapsules or the same molar concentration of dFdC as a control. After incubation for 1 h, 1.5 h, 2 h respectively, the culture medium was removed. The cells were washed with cold PBS for 3 times and then treated with 2 mL of 1% SDS lysis buffer for 10 min. The cells lysate was freeze-dried and dissolved by 200 L of the mixture of methanol and acetonitrile (the volume ratio of methanol and acetonitrile was 1:1). After filtration with 0.22 m membrane filter, the concentration of dFdC was determined by HPLC. Values were reported as the means for each triplicate sample. Means and corresponding standard deviations (mean ± SD) were shown as results. 2.8. Statistical data analysis Statistical data analysis was performed using the Student’s ttest. 2.9. Characterization and measurements 1 H NMR spectra were recorded for native gemcitabine, VES and VES-dFdC from a Bruker AvariceTM 400 NMR spectrometer using DMSO-d6 as solvent. For 1 H NMR measurement, concentration of the sample was 35 mg/mL. 13 C NMR spectra were recorded with the same spectrometer using acetone-d6 as solvent. The fourier transform infrared spectrometer (FTIR) data of native gemcitabine, VES and VES-dFdC were obtained from Nicolet Nexus 670 spectrometer. The samples were pressed into pellets with KBr.
3.1. The synthesis and characterization of VES-dFdC The synthesis scheme of VES-dFdC prodrug was shown as Fig. 1. This method was chosen due to the simple operation and high reaction efficiency. The 1 H NMR spectra of VES-dFdC prodrug, VES and dFdC were displayed in Fig. 2. Peaks belonged to VES and dFdC were labeled in the 1 H NMR spectrum of VES-dFdC. The proton signals of 3 ,5 -OH group of the pyrimidine ring of VES-dFdC (chemical shifts were at 6.2 ppm, 5.2 ppm respectively) were as same as that of dFdC. In addition, the absence of the peaks at 7.4 ppm belonging to N4 amino group of the pyrimidine ring of dFdC and the appearance of the peaks at 11.2 ppm assigning to amide group both confirmed that VES-dFdC was linked by the amide bond rather than the ester bond. The infrared spectrum of VES-dFdC prodrug, VES and dFdC were shown in Fig. 3. The disappearance of the peaks at 3400–3100 cm−1 assigned to the amino group and the appearance of the typical peaks at 1709, 1492, and 1245 cm−1 further confirmed that the carboxyl group of VES reacted with the amino group rather than the hydroxyl group of dFdC. 3.2. The CVC of VES-dFdC nanocapsules The CVC is defined as the concentration above which nanoscale aggregates can be spontaneously formed, and is one of the important characteristics of the amphiphilic compound. The VES-dFdC was inclined to form nanocapsules in aqueous media due to its amphiphilic property and its CVC was obtained by fluorospectrophotometer with pyrene as a probe. The plot of the intensity ratio I373 /I384 of the pyrene in emission spectrum against the logarithm of the concentration of VES-dFdC was shown in Fig. 4. When the concentration of the conjugate reaches the CVC, there is a sudden change of I373 /I384 in the
Fig. 1. Synthesis scheme of the VES-dFdC prodrug.
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Fig. 2.
1
H NMR of (A) dFdC, (B) VES, (C) VES-dFdC and (D) 13 C NMR of VES-dFdC.
fluorescence spectrum due to the transfer of pyrene from a polar environment to a non-polar environment caused by the formation of nanocapsules. The VES-dFdC conjugate showed a CVC of 0.004 mg/mL. The CVC of VES-dFdC was pretty low, indicating its good stability against dilution. 3.3. Characterization of VES-dFdC nanocapsules The size of the nanodrugs, one of significant properties, can highly affect their in vivo performance and pharmacokinetic properties. The size and zeta potential of the nanocapsules were measured by dynamic light scattering (DLS). As shown in Fig. 5A, the VES-dFdC nanocapsules showed an average size of ca. 107 nm with a narrow size distribution, suggesting its potential as a passive tumor-targeted capsule. The nanocapsules showed a negative surface potential of −33.4 mV, indicating its potential as a prolonged blood circulation time agent for that nanoparticles with positive surface charges would interplay with serum protein in
Fig. 3. FT-IR spectra of (A) dFdC and (B) VES-dFdC.
human bloods. The morphology of the VES-dFdC nanocapsules was observed by transmission electron microscopy (TEM). As shown in Fig. 5B, the nanocapsules had a less colored core surrounded by a dark membrane, which confirmed its vesicular structure. TEM micrograph revealed its nanoscale sizes of 45 nm, smaller than that measured by DLS, which might due to collapse of the nanocapsules during drying for the preparation of TEM specimen. 3.4. Drug release from VES-dFdC nanocapsules The controlled release properties of this prodrug were investigated under different pH values with or without cathepsin B, an amide hydrolase which is abundant in lysosomes. Fig. 6 showed the release profiles of VES-dFdC nanocapsules. At pH 7.4, less than 7% of dFdC were released within 12 h, which illustrates that the VES-dFdC nanocapsules can retain stability under physiological conditions (i.e., in the blood circulation), with low systemic toxicity. A similar release profile was obtained at a weakly acidic
Fig. 4. Plot of the ratios of the I373 /I384 value from the fluorescence spectra as a function of the logarithm concentration of the VES-dFdC nanocapsules.
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Fig. 5. (A) Size and size distributions of VES-dFdC nanocapsules measured by DLS; (B) Transmission electron microscopy (TEM) of VES-dFdC nanocapsules.
above, the VES-dFdC nanocapsules showed great potential as a prodrug for intracellular release of dFdC.
3.5. MTT assay
Fig. 6. In vitro release profiles of VES-dFdC nanocapsules at 37 ◦ C in (A) simulated lysosome environment (sodium acetate buffer pH 5 + cathepsin B), (B) PBS pH 7.4, and (C) sodium acetate buffer pH 5 (means ± SD, n = 3).
environment (pH 5). In contrast, the VES-dFdC nanocapsules showed an accelerated release in the presence of cathepsin B at pH 5 (as presented in endosomes and lysosomes) due to the degradation of amide bonds. More than 60% of dFdC was released in 4 h and nearly 100% of dFdC was released during 8 h. From the
The in vitro cytotoxicity of VES-dFdC nanocapsules was evaluated on human pancreatic cancer cell line (BxPC-3) via the MTT assay and representative concentration-growth inhibition bars showed the effects of treatment with free dFdC and VES-dFdC nanocapsules on the growth of BxPC-3 cells after 48 h. As shown in Fig. 7A, both free dFdC and VES-dFdC nanocapsules inhibited cell growth in a dose-dependent manner, whereas the latter was more toxic than the former especially when the concentration was higher than 2.9 M. Two reasons were thought to explain the increased cytotoxicity of VES-dFdC nanocapsules. First, nanocapusules have a similar structure of cell membrane, which make them easier than free dFdC to enter the tumor cells. This was confirmed in the following cellular uptake experiment. Second, the released VES, due to the hydrolysis of VES-dFdC prodrug by cathepsin B and cathepsin D, also play an important role in growth inhibition of the tumor cells especially at high concentration. As shown in Fig. 7B, when the concentration reached 47.5 M, VES revealed a wonderful activity to inhibit the growth of tumor cells and less than 40% of cells were viable. As a result, the mixture of VES and dFdC (1:1) exerted a much better inhibition of tumor cells than both of the individuals at the same concentration (47.5 M). Hence, both the released VES and
Fig. 7. Cell viability of (A) dFdC and VES-dFdC nanocapsules, (B) dFdC, VES and the mixture of VES and dFdC (mole ratio 1:1) at various concentrations against BxPC-3 cells line for 48 h (mean ± SD, n = 6, *P < 0.05).
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narrow size distribution. The negative zeta potential and the pretty low CVC both resulted in its remarkable colloidal stability. In addition, in contrast with free dFdC, VES-dFdC nanocapsules displayed a successful intracellular release of dFdC and more efficient uptake by the cancer cells, thus increased its cytotoxcity to tumor cells. Collectively, VES-dFdC nanocapsules suggested a great potential as prodrug for pancreatic tumor therapy. Acknowledgements The research work was supported by the International Science & Technology Cooperation Program of China, Ministry of Science and Technology of China (2013DFG32340), the EU-FP 7 project (MARINA 263215), and the National Natural Science Foundation of China (81171333). Appendix A. Supplementary data Fig. 8. Cellular uptake of dFdC and VES-dFdC nanocapsules (mean ± SD, n = 3).
dFdC might have contributed to the increased cytotoxcity of the VES-dFdC nanocapsules. 3.6. Cellular uptake of VES-dFdC nanocapsules The amount of uptake of the nanocapsules was measured by the HPLC, with free dFdC as a control. Fig. 8 revealed that very small uptake amount of the free dFdC was detected during 2 h incubation, while in the case of VES-dFdC nanocapsules, the maximum uptake amount of nanocapsules was more than 14% at 1.5 h, which is almost seventy times higher than the amount of free dFdC at the same time. However, the uptake amount of nanocapsules decreased from 14% to 10.4% when the incubation time increased to 2 h. It was thought that the decreased uptake of nanocapsules was attributed to the increased cell death induced by the release of dFdC in tumor cells. The dead cells were easily removed in treatment process resulted from the loss of adherence ability to the surface of culture plate. Together, these data suggested VES-dFdC nanocapsules passed through cell membrane much more easily than free dFdC, resulting in a higher anticancer efficiency. 4. Conclusions The nanocapsules based on the amphiphilic VES-dFdC prodrug displayed the capsule shapes with average size of ca. 107 nm and a
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. colsurfb.2015.02.028. References [1] S.B. Kaye, J. Clin. Oncol. 12 (1994) 1527–1531. [2] V. Heinemann, Y.Z. Xu, S. Chubb, et al., Mol. Pharmacol. 38 (1990) 567–572. [3] C.J. vanMoorsel, G. Veerman, A.M. Bergman, et al., Semin. Oncol. 24 (1997) S17–S23. [4] T. Neff, C.A. Blau, Exp. Hematol. 24 (1996) 1340–1346. [5] D.Y. Bouffard, J. Laliberte, R.L. Momparler, Biochem. Pharmacol. 45 (1993) 1857–1861. [6] J.R. Mackey, R.S. Mani, M. Selner, et al., Cancer Res. 58 (1998) 4349–4357. [7] J. Garcia-Manteiga, M. Molina-Arcas, F.J. Casado, et al., Cancer Res. 9 (2003) 5000–5008. [8] T. Oguri, H. Achiwa, H. Muramatsu, et al., Cancer Lett. 256 (2007) 112–119. [9] J. Spratlin, R. Sangha, D. Glubrecht, et al., Cancer Res. 10 (2004) 6956–6961. [10] M. Schirmer, A.P. Stegmann, F. Geisen, et al., Exp. Hematol. 26 (1998) 1223–1228. [11] P. Chen, P.Y. Chien, A.R. Khan, et al., Anticancer Drugs 17 (2006) 53–61. [12] L. Bildstein, C. Dubernet, V. Marsaud, et al., J. Control. Release 147 (2010) 163–170. [13] W.G. Chung, M.A. Sandoval, B.R. Sloat, et al., J. Control. Release 157 (2012) 132–140. [14] S. Turley, F.W. Funakoshi, J.M. Ruscetti, et al., Cell Growth Differ. 6 (1995) 655–663. [15] W. Yu, K. Israel, Q.Y. Liao, et al., Cancer Res. 59 (1999) 953–961. [16] K. Israel, W. Yu, B.G. Sanders, et al., Nutr. Cancer 36 (2000) 90–100. [17] J. Quin, D. Engle, A. Litwiller, et al., J. Surg. Res. 127 (2005) 139–143. [18] K.T. Barnett, F.D. Fokum, M.P. Malafa, J. Surg. Res. 106 (2002) 292–298. [19] P. Couvreur, B. Stella, L.H. Reddy, et al., Nano Lett. 6 (2006) 2544–2548.
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Enhanced cellular uptake and intracellular drug controlled release of VESylated gemcitabine prodrug nanocapsules Yanfen Fang a,1 , Fang Du a,b,1 , Yanyun Xu a , Haijing Meng a,c , Jin Huang a,d , Xiongwen Zhang a , Wei Lu a , Shiyuan Liu e , Jiahui Yu a,∗ a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, College of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, PR China b Institute of Chemistry and Biochemistry, Free University Berlin, Takustr 3, 14195 Berlin, Germany c Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw 01-224, Poland d College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, PR China e Department of Diagnostic Imaging, ChangZheng Hospital, Shanghai 200003, PR China
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Article history: Received 3 November 2014 Received in revised form 15 January 2015 Accepted 13 February 2015 Available online xxx Keywords: VESylated gemcitabine Nanocapsules Intracellular drug controlled release Enhanced cellular uptake
a b s t r a c t Gemcitabine, 2 ,2 -difluoro-2 -deoxycytidine (dFdC), is the first-line antitumor agent in the treatment of pancreatic tumors. However, it possesses certain drawbacks, such as poor biological half-life resulted from rapid metabolism and the induction of resistance, leading to its restricted therapeutic potential. With the purpose of overcoming the above drawbacks, we developed a novel VESylated gemcitabine (VES-dFdC) prodrug by coupling the N4 -amino group of the pyrimidine ring of dFdC to the carboxylic group of vitamin E succinate (VES). The resulting amphiphilic compound could protect the N4 -amino group of the pyrimidine ring of dFdC from being degraded by cytidine deaminase. What is more, the prodrug was able to form nanocapsules in aqueous media (similar to the structure of cytomembrane), confirmed by transmission electron microscope (TEM). Their average particle size is about 107 nm with zeta potential of −33.4 mV measured by dynamic light scattering (DLS). VES-dFdC nanocapsules showed accelerated accumulative drug release profile in simulated lysosome environment (sodium acetate buffer pH 5 + cathepsin B, an enzyme in lysosome), due to the easily hydrolyzed property of amide bond by cathepsin B, while rather stable in PBS (pH 7.4) or sodium acetate buffer (pH 5.0) without cathepsin B, indicating their enhanced intracellular drug controlled release manner. Besides, VES-dFdC prodrug nanocapsules showed enhanced cellular uptake ability, and the amount of cellular uptake of the nanocapsules by the pancreatic cancer cell line BxPC-3 is seventy times higher than that of native gemcitabine in the first 1.5 h. Compared with free gemcitabine, VES-dFdC nanocapsules showed essentially increased growth inhibition activity against BxPC-3 cells, indicating its great potential as prodrug for pancreatic tumor therapy with improved antitumor activity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Gemcitabine, 2 ,2 -difluoro-2 -deoxycytidine (dFdC), is a pyrimidine nucleoside analog anticancer agent. It is the first-line treatment against pancreatic cancer, and often used in combination therapy for non-small lung, ovarian and breast tumors [1–3]. However, dFdC is easily metabolized intracellularly and extracellularly by cytidine deaminase (CDA) into the chemotherapeutically inactive uracil derivative, leading to a very short plasma half-life
∗ Corresponding author. Tel.: +86 21 6223 7026; fax: +86 21 6223 7026. E-mail address: [email protected] (J. Yu). 1 These authors contributed equally to this work.
[4,5]. Since dFdC is a hydrophilic compound, it requires membrane proteins, such as human equilabrative nucleoside transporter-1 (hENT1) to transport it into the cells [6]. Therefore, tumor cells that have decreased expression of nucleoside transporters are resistant to dFdC [7]. Moreover, the resistance to dFdC is the major issue in its clinical application for the treatment of pancreatic cancer [8,9]. Great efforts have been made to overcome dFdC resistance. Schirmer et al. synthesized the amino acid ester prodrugs of dFdC, which were not as sensitive as dFdC to deamination by CDA [10]. To enhance the cellular uptake of dFdC, amphiphilic prodrugs of dFdC have been constructed to circumvent this specific resistance pathway since these compounds are expected to passively diffuse across cell membranes. The uptake of dFdC-cardiolipin conjugate was proven transporter-independent, which was consistent with
http://dx.doi.org/10.1016/j.colsurfb.2015.02.028 0927-7765/© 2015 Elsevier B.V. All rights reserved.
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its passive diffusion through the plasma membrane [11]. Recently, squalenoylated dFdC prodrugs were synthesized, and formulated into nanoparticles. These nanoparticles were more cytotoxic than gemcitabine in two dFdC resistance cell lines [12]. Similarly, Cui et al. discovered that the 4-(N)-stearoyl dFdC solid lipid nanoparticles were more cytotoxic than dFdC in dFdC resistance related to ribonucleotide reductase M1 over-expression [13]. Vitamin E is one of the lipophilic vitamins. Vitamin E succinate (VES) possesses good antitumor activity against a wide variety of tumors by the induction of cell apoptosis [14–17], while shows little toxicity against normal cells or tissue [18]. Herein, VES-dFdC prodrug was developed by coupling the N4 -amino group of the pyrimidine ring of dFdC to the carboxylic group of VES. The structure of the prodrug was characterized by 1 H NMR and FT IR. The VES-dFdC prodrug nanocapsules were fabricated, and their physicochemical properties, such as CAC, size and drug accumulative release were investigated. And then, cytotoxicity and cellular uptake ability were evaluated in detail. 2. Materials and methods 2.1. Materials Gemcitabine and isobutyl chloroformate were purchased from Alfa Aesar. Vitamin E succinate was bought from Aladdin Chemistry Co. Lid. Triethylamine (TEA), dimethyl formamide (DMF), and tetrahydrofuran (THF) were obtained from Sinopharm Chemical Reagent Co. Ltd. TEA was dried with 4 A˚ molecular sieve and redistilled before use. DMF and THF were dried over calcium hydride and were distilled in the presence of fresh calcium hydride just before use. 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and cathepsin B were purchased from Sigma–Aldrich. 2.2. Cell line and culture Human pancreatic cancer cell line (BxPC-3, adherent cell) were purchased from Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences. BxPC-3 cells were cultured in RPMI 1640 (Gibco BRL, Paris, France), supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT), 1% penicillin and streptomycin. BxPC-3 cells were incubated at 37 ◦ C in humidified 5% CO2 atmosphere. When the cell confluence of 90% was reached, they were routinely trypsinized and subcultured. 2.3. Synthesis of VES-dFdC The VES-dFdC prodrug was synthesized as previously described with slightly modification [19]. Briefly, triethylamine (0.17 mL, 1.2 mmol) was added to a stirred solution of vitamin E succinate (0.53 g, 1.0 mmol) in 5 mL of anhydrous THF. The mixture was cooled to −15 ◦ C, and a solution of isobutyl chloroformate (0.13 mL, 1.2 mmol) in 5 mL of anhydrous THF was added dropwise. The mixture was stirred at −15 ◦ C for 30 min and a solution of gemcitabine hydrochloride (0.300 g, 1.0 mmol) and triethylamine (0.17 mL, 1.2 mmol) in anhydrous DMF (5 mL) was added dropwise to the reaction mixture at the same temperature. The reaction mixture was stirred for 72 h at room temperature, and then concentrated in vacuo. Aqueous sodium hydrogen carbonate was added, and the mixture was extracted with ethyl acetate (3 mL × 50 mL). The combined extracts were washed with water, dried over anhydrous Na2 SO4 , and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel eluting with 1–4% methanol in dichloromethane to give pure VES-dFdC as white solid (yield: 53%). IR (neat, cm−1 ) 3500–3150, 2926, 2856, 1709, 1656, 1492, 1245; 1 H NMR (400 MHz, DMSO-d6 ) 11.15 (s, 1 H,
NHCO), 8.26 (d, 1 H, H6), 7.26 (d, 1 H, H5), 6.32 (d, 1 H, OH3), 6.18 (t, 1 H, H1), 5.29 (t, 1 H, OH5), 4.18 (m, 1 H, H3), 3.86–3.68 (m, 3 H, H4 and H5), 2.91 (m, 2 H), 2.85 (m, 2 H), 2.56 (m, 2 H), 2.00 (s, 3 H), 1.92 (s, 3 H), 1.90 (s, 3 H), 1.75 (m, 2 H), 1.02 (m, 3 H), 0.84 (s, 6 H), 0.82 (s, 6 H), 0.81 (s, 3 H); 13 C NMR (100 M, acetone-d6 ) ␦ 173.4-C25 , 171.71C22 , 164.0-C17 , 155.63-C19 , 149.98-C30 , 145.87-C15 , 141.82-C32 , 127.68-C28 , 126.14-C27 , 123.93-C31 , 123.16-C29 , 118.31-C2 , 96.99C14 , 82.49-C13 , 75.75-C39 , 70.13-C4 , 69.79-C1 , 60.21-C10 , 40.14-C40 , 38.24-C54 , 38.18-C45 , 38.11-C50 , 38.03-C52 , 33.49-C43 , 32.55-C44 , 31.97-C51 , 30.50-C38 , 30.31-C24 , 29.61-C23 , 29.32-C53 , 28.84-C49 , 28.71-C38 , 25.55-C57 , 25.15-C58 , 23.01-C42 , 21.70-C37 , 21.14-C44 , 20.13-C53 , 13.20-C35 , 12.32-C33 , 12.02-C34 . MS (ESI): m/z (%): 776 [M+]. 2.4. Nanocapsule fabrication and the critical vesicle concentration The VES-dFdC (6 mg) conjugate was dissolved in 2 mL of acetone, and stirred at room temperature for 1 h, then the acetone solution was added dropwise into 6 mL of ultrapure water under constant stirring. After then, the solution was loaded into a dialysis tube (MWCO 3500) and dialyzed against 8 L (2 L × 4) of deionized water in the dark for 16 h. The critical vesicle concentration (CVC) was determined using pyrene as a fluorescence probe. The concentration of VES-dFdC varied from 7.5 × 10−5 to 1 mg/mL with fixed pyrene concentration of 6 × 10−7 mol/L. The fluorescence spectra were measured on F-4500 fluorescence spectrophotometer (Hitchi F-4500) with the excitation wavelength of 335 nm. The I373 /I384 of fluorescence intensity ratio in the emission spectra of pyrene was analyzed for the calculation of CVC. 2.5. Release of dFdC from VES-dFdC nanocapsules The rate of release of dFdC from VES-dFdC prodrug was measured with RP-HPLC system of Agilent 1200 (Agilent Technologies Inc. Shanghai Branch) using a Zorbax Eclipse XDB-C18 column (5 m, 4.6 mm × 250 mm) at 30 ◦ C. The release of the drug from VES-dFdC was performed by dissolving the conjugate 1 mg/mL in DMSO, then 30 L of the DMSO solution was dispersed into 1 mL of phosphate buffer saline (PBS) (pH 7.4), sodium acetate buffer (pH 5.0) and also sodium acetate buffer (pH 5.0) in the presence of 10 U cathepsin B. Briefly, the samples were kept in a THZ-C isothermal shaker at 37 ◦ C and 150 rpm. At predetermined time point, 100 L of solution was withdrawn from the samples for HPLC analysis using pure methanol as the mobile phase. The flow rate of the mobile phase was 1 mL/min. The Agilent 1200 Uv/vis detector was set at 248 nm. The release percentage of dFdC was calculated from the ratio of peaks area assigned to free dFdC and VES-dFdC. Experiments were carried out up to 96 h in triplicates. 2.6. In vitro tumor cell growth inhibition activity of VES-dFdC nanocapsules In vitro tumor cell growth inhibition activity of VES-dFdC nanocapsules was determined by evaluation of the viability of pancreatic cancer cell line (BxPC-3) by MTT method. Cells were seeded in 96-well plates at an initial density of 4 × 103 cells/well in 200 L RPMI-1640 culture medium and incubated for 18–24 h to reach 80% confluence at the time of treatment. Culture medium was replaced with 100 L fresh media containing various amounts of VES-dFdC conjugate, dFdC and VES (0, 0.15, 1.5, 2.9, 5.9, 11.8, 23.7 and 47.5 M). Cells were incubated for 48 h. And then, 10 L of MTT solution (0.5 mg/mL) was added into the wells. After further incubation for 4 h in incubator, 100 L of DMSO was added to each well to replace the culture medium and dissolve the insoluble
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formazan-containing crystals. The optical density was measured at 570 nm using an automatic BIO-TEK microplate reader (Powerwave XS, USA), and the cell viability was calculated from following equation: Cell viability (%) =
ODsample ODcontrol
× 100%
(1)
Where ODsample represents an OD value from a well treated with samples and ODcontrol from a well treated with PBS (0.01 M, pH 7.4) buffer only. Values were reported as the means for each triplicate sample. Means and corresponding standard deviations (mean ± SD) were shown as results.
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Fluorescence measurement was performed on a Hitachi F-4500 Fluorescence Spectrophotometer, samples were dissolved in pure water, and pyrene was used as the fluorescent probe. The particle sizes, size distributions and zeta () potentials of nanocapsules were measured by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, UK). Morphological evaluation of the nanocapsules was obtained with a Philips CM 120 transmission electron microscope (TEM), working at 60-kV accelerating voltage. A 10 L of capsule solution of 10−1 mg/mL was carefully dropped onto clean copper grids, and then dried at room temperature for 30 min before imaging on the microscope. The average size of the capsules was determined from the TEM images and the scale bars were sharpened with Photoshop 8.0 software.
2.7. In vitro evaluation of cellular uptake 3. Results and discussion The amount of the uptake of VES-dFdC nanocapsules by the BxPC-3 cells was measured by the HPLC with free dFdC as a control in this experiment. Cells were seeded in 6-wells plate at an initial density of 2.5 × 105 cells per well in 2 mL of culture medium. After overnight incubation, the old culture medium was displaced by 2 mL of fresh medium which contains VES-dFdC nanocapsules or the same molar concentration of dFdC as a control. After incubation for 1 h, 1.5 h, 2 h respectively, the culture medium was removed. The cells were washed with cold PBS for 3 times and then treated with 2 mL of 1% SDS lysis buffer for 10 min. The cells lysate was freeze-dried and dissolved by 200 L of the mixture of methanol and acetonitrile (the volume ratio of methanol and acetonitrile was 1:1). After filtration with 0.22 m membrane filter, the concentration of dFdC was determined by HPLC. Values were reported as the means for each triplicate sample. Means and corresponding standard deviations (mean ± SD) were shown as results. 2.8. Statistical data analysis Statistical data analysis was performed using the Student’s ttest. 2.9. Characterization and measurements 1 H NMR spectra were recorded for native gemcitabine, VES and VES-dFdC from a Bruker AvariceTM 400 NMR spectrometer using DMSO-d6 as solvent. For 1 H NMR measurement, concentration of the sample was 35 mg/mL. 13 C NMR spectra were recorded with the same spectrometer using acetone-d6 as solvent. The fourier transform infrared spectrometer (FTIR) data of native gemcitabine, VES and VES-dFdC were obtained from Nicolet Nexus 670 spectrometer. The samples were pressed into pellets with KBr.
3.1. The synthesis and characterization of VES-dFdC The synthesis scheme of VES-dFdC prodrug was shown as Fig. 1. This method was chosen due to the simple operation and high reaction efficiency. The 1 H NMR spectra of VES-dFdC prodrug, VES and dFdC were displayed in Fig. 2. Peaks belonged to VES and dFdC were labeled in the 1 H NMR spectrum of VES-dFdC. The proton signals of 3 ,5 -OH group of the pyrimidine ring of VES-dFdC (chemical shifts were at 6.2 ppm, 5.2 ppm respectively) were as same as that of dFdC. In addition, the absence of the peaks at 7.4 ppm belonging to N4 amino group of the pyrimidine ring of dFdC and the appearance of the peaks at 11.2 ppm assigning to amide group both confirmed that VES-dFdC was linked by the amide bond rather than the ester bond. The infrared spectrum of VES-dFdC prodrug, VES and dFdC were shown in Fig. 3. The disappearance of the peaks at 3400–3100 cm−1 assigned to the amino group and the appearance of the typical peaks at 1709, 1492, and 1245 cm−1 further confirmed that the carboxyl group of VES reacted with the amino group rather than the hydroxyl group of dFdC. 3.2. The CVC of VES-dFdC nanocapsules The CVC is defined as the concentration above which nanoscale aggregates can be spontaneously formed, and is one of the important characteristics of the amphiphilic compound. The VES-dFdC was inclined to form nanocapsules in aqueous media due to its amphiphilic property and its CVC was obtained by fluorospectrophotometer with pyrene as a probe. The plot of the intensity ratio I373 /I384 of the pyrene in emission spectrum against the logarithm of the concentration of VES-dFdC was shown in Fig. 4. When the concentration of the conjugate reaches the CVC, there is a sudden change of I373 /I384 in the
Fig. 1. Synthesis scheme of the VES-dFdC prodrug.
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Fig. 2.
1
H NMR of (A) dFdC, (B) VES, (C) VES-dFdC and (D) 13 C NMR of VES-dFdC.
fluorescence spectrum due to the transfer of pyrene from a polar environment to a non-polar environment caused by the formation of nanocapsules. The VES-dFdC conjugate showed a CVC of 0.004 mg/mL. The CVC of VES-dFdC was pretty low, indicating its good stability against dilution. 3.3. Characterization of VES-dFdC nanocapsules The size of the nanodrugs, one of significant properties, can highly affect their in vivo performance and pharmacokinetic properties. The size and zeta potential of the nanocapsules were measured by dynamic light scattering (DLS). As shown in Fig. 5A, the VES-dFdC nanocapsules showed an average size of ca. 107 nm with a narrow size distribution, suggesting its potential as a passive tumor-targeted capsule. The nanocapsules showed a negative surface potential of −33.4 mV, indicating its potential as a prolonged blood circulation time agent for that nanoparticles with positive surface charges would interplay with serum protein in
Fig. 3. FT-IR spectra of (A) dFdC and (B) VES-dFdC.
human bloods. The morphology of the VES-dFdC nanocapsules was observed by transmission electron microscopy (TEM). As shown in Fig. 5B, the nanocapsules had a less colored core surrounded by a dark membrane, which confirmed its vesicular structure. TEM micrograph revealed its nanoscale sizes of 45 nm, smaller than that measured by DLS, which might due to collapse of the nanocapsules during drying for the preparation of TEM specimen. 3.4. Drug release from VES-dFdC nanocapsules The controlled release properties of this prodrug were investigated under different pH values with or without cathepsin B, an amide hydrolase which is abundant in lysosomes. Fig. 6 showed the release profiles of VES-dFdC nanocapsules. At pH 7.4, less than 7% of dFdC were released within 12 h, which illustrates that the VES-dFdC nanocapsules can retain stability under physiological conditions (i.e., in the blood circulation), with low systemic toxicity. A similar release profile was obtained at a weakly acidic
Fig. 4. Plot of the ratios of the I373 /I384 value from the fluorescence spectra as a function of the logarithm concentration of the VES-dFdC nanocapsules.
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Fig. 5. (A) Size and size distributions of VES-dFdC nanocapsules measured by DLS; (B) Transmission electron microscopy (TEM) of VES-dFdC nanocapsules.
above, the VES-dFdC nanocapsules showed great potential as a prodrug for intracellular release of dFdC.
3.5. MTT assay
Fig. 6. In vitro release profiles of VES-dFdC nanocapsules at 37 ◦ C in (A) simulated lysosome environment (sodium acetate buffer pH 5 + cathepsin B), (B) PBS pH 7.4, and (C) sodium acetate buffer pH 5 (means ± SD, n = 3).
environment (pH 5). In contrast, the VES-dFdC nanocapsules showed an accelerated release in the presence of cathepsin B at pH 5 (as presented in endosomes and lysosomes) due to the degradation of amide bonds. More than 60% of dFdC was released in 4 h and nearly 100% of dFdC was released during 8 h. From the
The in vitro cytotoxicity of VES-dFdC nanocapsules was evaluated on human pancreatic cancer cell line (BxPC-3) via the MTT assay and representative concentration-growth inhibition bars showed the effects of treatment with free dFdC and VES-dFdC nanocapsules on the growth of BxPC-3 cells after 48 h. As shown in Fig. 7A, both free dFdC and VES-dFdC nanocapsules inhibited cell growth in a dose-dependent manner, whereas the latter was more toxic than the former especially when the concentration was higher than 2.9 M. Two reasons were thought to explain the increased cytotoxicity of VES-dFdC nanocapsules. First, nanocapusules have a similar structure of cell membrane, which make them easier than free dFdC to enter the tumor cells. This was confirmed in the following cellular uptake experiment. Second, the released VES, due to the hydrolysis of VES-dFdC prodrug by cathepsin B and cathepsin D, also play an important role in growth inhibition of the tumor cells especially at high concentration. As shown in Fig. 7B, when the concentration reached 47.5 M, VES revealed a wonderful activity to inhibit the growth of tumor cells and less than 40% of cells were viable. As a result, the mixture of VES and dFdC (1:1) exerted a much better inhibition of tumor cells than both of the individuals at the same concentration (47.5 M). Hence, both the released VES and
Fig. 7. Cell viability of (A) dFdC and VES-dFdC nanocapsules, (B) dFdC, VES and the mixture of VES and dFdC (mole ratio 1:1) at various concentrations against BxPC-3 cells line for 48 h (mean ± SD, n = 6, *P < 0.05).
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narrow size distribution. The negative zeta potential and the pretty low CVC both resulted in its remarkable colloidal stability. In addition, in contrast with free dFdC, VES-dFdC nanocapsules displayed a successful intracellular release of dFdC and more efficient uptake by the cancer cells, thus increased its cytotoxcity to tumor cells. Collectively, VES-dFdC nanocapsules suggested a great potential as prodrug for pancreatic tumor therapy. Acknowledgements The research work was supported by the International Science & Technology Cooperation Program of China, Ministry of Science and Technology of China (2013DFG32340), the EU-FP 7 project (MARINA 263215), and the National Natural Science Foundation of China (81171333). Appendix A. Supplementary data Fig. 8. Cellular uptake of dFdC and VES-dFdC nanocapsules (mean ± SD, n = 3).
dFdC might have contributed to the increased cytotoxcity of the VES-dFdC nanocapsules. 3.6. Cellular uptake of VES-dFdC nanocapsules The amount of uptake of the nanocapsules was measured by the HPLC, with free dFdC as a control. Fig. 8 revealed that very small uptake amount of the free dFdC was detected during 2 h incubation, while in the case of VES-dFdC nanocapsules, the maximum uptake amount of nanocapsules was more than 14% at 1.5 h, which is almost seventy times higher than the amount of free dFdC at the same time. However, the uptake amount of nanocapsules decreased from 14% to 10.4% when the incubation time increased to 2 h. It was thought that the decreased uptake of nanocapsules was attributed to the increased cell death induced by the release of dFdC in tumor cells. The dead cells were easily removed in treatment process resulted from the loss of adherence ability to the surface of culture plate. Together, these data suggested VES-dFdC nanocapsules passed through cell membrane much more easily than free dFdC, resulting in a higher anticancer efficiency. 4. Conclusions The nanocapsules based on the amphiphilic VES-dFdC prodrug displayed the capsule shapes with average size of ca. 107 nm and a
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. colsurfb.2015.02.028. References [1] S.B. Kaye, J. Clin. Oncol. 12 (1994) 1527–1531. [2] V. Heinemann, Y.Z. Xu, S. Chubb, et al., Mol. Pharmacol. 38 (1990) 567–572. [3] C.J. vanMoorsel, G. Veerman, A.M. Bergman, et al., Semin. Oncol. 24 (1997) S17–S23. [4] T. Neff, C.A. Blau, Exp. Hematol. 24 (1996) 1340–1346. [5] D.Y. Bouffard, J. Laliberte, R.L. Momparler, Biochem. Pharmacol. 45 (1993) 1857–1861. [6] J.R. Mackey, R.S. Mani, M. Selner, et al., Cancer Res. 58 (1998) 4349–4357. [7] J. Garcia-Manteiga, M. Molina-Arcas, F.J. Casado, et al., Cancer Res. 9 (2003) 5000–5008. [8] T. Oguri, H. Achiwa, H. Muramatsu, et al., Cancer Lett. 256 (2007) 112–119. [9] J. Spratlin, R. Sangha, D. Glubrecht, et al., Cancer Res. 10 (2004) 6956–6961. [10] M. Schirmer, A.P. Stegmann, F. Geisen, et al., Exp. Hematol. 26 (1998) 1223–1228. [11] P. Chen, P.Y. Chien, A.R. Khan, et al., Anticancer Drugs 17 (2006) 53–61. [12] L. Bildstein, C. Dubernet, V. Marsaud, et al., J. Control. Release 147 (2010) 163–170. [13] W.G. Chung, M.A. Sandoval, B.R. Sloat, et al., J. Control. Release 157 (2012) 132–140. [14] S. Turley, F.W. Funakoshi, J.M. Ruscetti, et al., Cell Growth Differ. 6 (1995) 655–663. [15] W. Yu, K. Israel, Q.Y. Liao, et al., Cancer Res. 59 (1999) 953–961. [16] K. Israel, W. Yu, B.G. Sanders, et al., Nutr. Cancer 36 (2000) 90–100. [17] J. Quin, D. Engle, A. Litwiller, et al., J. Surg. Res. 127 (2005) 139–143. [18] K.T. Barnett, F.D. Fokum, M.P. Malafa, J. Surg. Res. 106 (2002) 292–298. [19] P. Couvreur, B. Stella, L.H. Reddy, et al., Nano Lett. 6 (2006) 2544–2548.
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