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Kinetics and Efficiency of a MethylCarboxylated 5-Fluorouracil-Bovine Serum Albumin Adduct for Targeted Deliverya Michael J. Koziol, Torsten K. Sievers, Kathrin Smuda, Yu Xiong, € ller, Felix Wojcik, Axel Steffen, Margitta Dathe, Angelika Mu €umler* Radostina Georgieva, Hans Ba

5-Fluorouracil (5-FU) is a clinically well-established anti-cancer drug effectively applied in chemotherapy, mainly for the treatment of breast and colorectal cancer. Substantial disadvantages are adverse effects, arising from serious damage of healthy tissues, and shortcoming pharmacokinetics due to its low molecular weight. A promising approach for improvement of such drugs is their coupling to suitable carriers. Here, a 5-FU adduct, 5-fluorouracil acetate (FUAc) is synthesized and covalently coupled to bovine serum albumin (BSA) as model carrier molecule. On average, 12 molecules FUAc are bound to one BSA. Circular dichriosm (CD)-spectra of BSA and FUAc-BSA are identical, suggesting no significant conformational differences. FUAc-BSA is tested on T-47D and MDA-MB-231 breast cancer cells. Proliferation inhibition of membrane albuminbinding protein (mABP)-expressing T-47D cells by FUAc-BSA is similar to that of 5-FU and only moderate for MDA-MB-231 cells that lack such expression. Therefore, a crucial role of mABP expression in effective cell growth inhibition by FUAc-BSA is assumed.

1. Introduction Cancer belongs to the major cause of death worldwide. According to the World Health Organization (WHO), cancer was responsible for about 7.6 million deaths (13% overall) in M. J. Koziol, K. Smuda, Dr. Y. Xiong, A. M€ uller, A. Steffen, €umler Dr. R Georgieva, Prof. H. Ba Institute of Transfusion Medicine, Center for Tumor Medicine,
- Universita €tsmedizin Berlin Charit
eplatz 1, 10117 Berlin, Charite Germany E-mail: [email protected] a Supporting Information is available from the Wiley Online Library or from the author.

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2008. Worldwide this number is estimated to reach 13.1 million in 2030.[1] Besides surgical and radiation therapies, chemotherapy is one of the essential cancer therapies.[2] 5-Fluorouracil (5-FU) is a cytostatic agent which belongs to the group of Dr. T. K. Sievers, F. Wojcik Max-Planck-Institute of Colloids and Interfaces, Wissenschaftspark Golm 14424 Potsdam, Germany Dr. M. Dathe Leibniz Institute of Molecular Pharmacology Robert-Roessle-Str. 10, 13125 Berlin, Germany Dr. R. Georgieva Department of Medical Physics, Biophysics and Image Diagnostics, Medical Faculty, Trakia University 6000 Stara Zagora, Bulgaria

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DOI: 10.1002/mabi.201300363

Kinetics and Efficiency of Methyl-Carboxylated 5-Fluorouracil-BSA Adduct www.mbs-journal.de

anti-metabolites. In combination with other drugs, it is widely used for treatment of many cancer types like colorectal, breast, pancreatic cancers, as well as head and neck cancers.[3–7] The cytostatic effect results from incorporation of the drug into DNA and RNA. Moreover, it inhibits the cellular enzyme thymidylate-synthase, leading to DNA and RNA strand breaks.[5] Disadvantages of cancer treatment with small molecules in general and especially with 5-FU arise from the capability of these molecules to enter nearly all body tissues. 5-FU does not accumulate only in cancer cells but is almost omnipresent in all, also healthy tissues.[8] Therefore, the therapy with 5-FU includes side effects like mucositis, myelosuppression, diarrhea and neurotoxicity.[9] Additionally, the fast decrease of 5-FU concentration in patients’ blood plasma (half-life approximately 10–20 min) and the excretion of 60–90% of 5-FU metabolites within 24 h indicate a major pharmacokinetic shortcoming.[10] Hence, a modification of 5-FU that aims to improve the pharmacokinetic efficiency and to reduce the adverse effects in 5-FU therapy would provide a major step forward in current cancer therapy. Such a modification may include chemical derivation of 5-FU or a 5-FU-loaded carrier. However, the modified 5-FU should meet several criteria: i) preferably similar toxicity as pure 5-FU to tumor cells, ii) reduced adverse effects, iii) increased plasma half-life for longer administration intervals. These criteria can be fulfilled by a carrier which allows for extending the intravascular circulation time and simultaneously delivering the cytostatic agent only to tumor cells, as in targeted therapy. Many carrier systems, such as microand nanoparticles, liposomes, micelles, or even red blood cells, are currently under consideration.[8,11] A promising strategy is also the use of macromolecules. Albumin, for example, would meet all these requirements as a carrier. With a concentration of about 35–50 mg mL
1, it represents 50–70% of human plasma proteins and is, therefore, very common in the human organism. Human serum albumin (HSA) has a half-life in the plasma of approximately 19 d in humans.[12,13] A drug coupled to albumin would, hence, exhibit an extended plasma half-life and as consequence an extension in duration of effect.[4] In addition, it was shown that tumor cells specifically take up plasma proteins, especially albumin, as these proteins represent a source of nitrogen and energy for these cells. Albumin can leave the capillary system by transcytosis to the extracellular space via a 60 kDa glycoprotein (gp60) and, therefore, occurs nearly everywhere in the human body.[12,14] Plasma proteins, and particularly albumin, are endocytosed by tumor cells and subsequently degraded in lysosomes. The resulting free amino acids are transferred into the cytoplasm for further usage.[12] Hence, an albumin-coupled cytostatic agent would probably attain the cytoplasm as well. A similar concept already led to the development of the clinically approved, targeted delivery drug

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nabTM-Paclitaxel, an albumin-bound taxane, which is used in metastatic breast cancer therapy.[4] Additionally, methotrexate covalently coupled to HSA (MTX-HSA) is under consideration in clinical studies.[15,16] However, only one molecule MTX was bound per HSA to retain the native protein structure which is necessary for tumor targeting.[17,18] Inspired by the successful targeted delivery of cytostatics, as in nab-Paclitaxel and MTX-HSA, we aimed to couple 5-FU to albumin. A Korean workgroup already described a high plasma-stability for acetate-modified 5-FU (FUAc) covalently coupled to HSA in in vivo and in vitro experiments.[19,20] However, the chemical synthesis and the amount of bound FUAc-molecules per albuminmolecule are not mentioned in literature. Cytostatic effects are not described, either. Therefore, we developed a procedure to couple FUAc covalently to the model substance bovine serum albumin (BSA) and investigated changes in BSA structure and the cytostatic potential in in vitro experiments.

2. Experimental Section 2.1. Materials 5-FU (MW ¼ 130.08), BSA (MW ca. 66 000), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), sodium hydrogencarbonate (NaHCO3), sodium dodecyl sulfate (SDS), phosphate buffered saline (PBS, 0.01 M), potassium hydroxide (KOH), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (MW ¼ 191.7), N-hydroxysuccinimide (NHS) (MW ¼ 115.1), 2-(N-morpholino)ethanesulfonic acid (MES) (MW ¼ 195.2) and bovine serum albumin–fluorescein isothiocyanate conjugate (FITC-BSA) (MWBSA ca. 66 000, MWFITC ¼ 389) were purchased from Sigma-Aldrich (Germany). Chloroacetic acid (MW ¼ 94.5), hydrochloric acid (37%) and acetic acid were purchased from J.T. Baker (Deventer, Holland). Trypsin-ethylenediaminetetraacectic acid (Trypsin-EDTA, 0.25%) was purchased from Life Technologies GmbH (Darmstadt, Germany). All chemicals were used without further purification. In the synthesis, water from a Millipore filter system (aurium 611DI from Sartorius AG, Germany) with a conductivity of 0.055 mS cm
1 was used.

2.2. Synthesis of 2-(5-Fluoro-2,4-dioxo-3,4dihydropyrimidine-1(2H)-yl)acetic Acid (5-Fluorouracil Acetic Acid, FUAc) 5-FUAc was synthesized from 5-FU based on a procedure in the literature[21] as follows. 5-FU (1.73 g, 13.3  10
3 mol) was dissolved in 11 mL water containing KOH (1.73 g, 26.6  10
3 mol). Chloroacetic acid (1.25 g, 13.3  10
3 mol) was dissolved in 5 mL water. The chloroacetic acid solution was poured into the 5-FU solution and the mixture was stirred under ambient conditions for 15 min. A white precipitate was formed, which was dissolved by gradual addition of solid potassium hydroxide until dissolution was just

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achieved (pH  10). Afterwards, the mixture was stirred for another 15 min and the pH was kept at 10 to avoid further precipitation. The clear solution was heated to 100 8C and boiled under reflux for 2.5 h. After cooling down to room temperature, pH was adjusted to 2 by dropwise addition of concentrated hydrochloric acid (10 M, 36% HCl). Importantly, the pH has to be adjusted carefully, because lower or higher pH values hinder the crystallization of FUAc. The solution was kept at 4 8C for 1 h and the pH was readjusted to 2. Afterwards, the solution was left for 24 h at 4 8C to allow for crystallization of FUAc in transparent, fine needles, which slowly began to form after the pH was readjusted. The solid FUAc was separated from the supernatant after centrifugation and dissolved in saturated, aqueous NaHCO3 (4 mL, just enough to obtain a clear solution). Afterwards, FUAc was again crystallized at 4 8C by adjusting the pH to 2 with concentrated hydrochloric acid (vide supra), separated from the supernatant, and dried under low pressure at 0.1 mbar and 30 8C to yield pure FUAc as a white powder. Notably, all supernatant solutions yielded additional FUAc, if kept for 6–12 months at 4 8C. However, this additional amount of product has not been regarded in the yield calculation. Yield: 745 mg (3.96  10
3 mol, MW ¼ 188.1), 29.8%. 1H-NMR (DMSO-d6, d): 13.23 (HOOC, 1H, s), 11.92 (H3, 1H, d, J4 ¼ 5.1 Hz), 8.08 (H6, 1H, d, J3 ¼ 6.8 Hz), 4.36 (H1‘, 2H, s). 13C-NMR broad band 1H decoupled (DMSO-d6, d): 169.43 (C2‘, 1C, s), 157.63 (C4, 1C, d, J2 ¼ 25.78 Hz), 149.82 (C2, 1C, s), 139.47 (C5, 1C, d, J1 ¼ 228.92 Hz), 130.69 (C6, 1C, d, J2 ¼ 33.93 Hz), 48.84 (C1‘, 1C, s). 19F-NMR (DMSO-d6, d):
170.15 ppm (F5, 1F, dvd, J3 ¼ 6.4 Hz, J4 ¼ 4.7 Hz). 1H-, 13C-, and 19F-NMR spectra were recorded on a DMX400 by Bruker (Rheinstetten, Germany) in DMSO-d6 unless otherwise noted.

2.3. Binding of FUAc to BSA The covalent binding of FUAc to BSA was performed according to the published EDC/NHS-method with some modifications.[22] BSA (300 mg,  4,55  10
6 mol, MW  66 000) was dissolved in 12 mL buffer (6 mL PBS þ 6 mL water). FUAc (300 mg, 1.6  10
3 mol, MW ¼ 188.1) and NaCl (300 mg) were dissolved in 12 mL water and pH was adjusted to 7 with 1 M NaOH. MES (300 mg, 1.2  10
3 mol) was added to the FUAc solution. EDC (600 mg, 3.13  10
3 mol) and NHS (900 mg, 7.82  10
3 mol) were dissolved in 2 mL water and immediately poured into the FUAc solution (vide supra) yielding a molar ratio of 1.96:1 (EDC:FUAc). The mixture was stirred at room temperature for 20 min and pH was adjusted afterwards with 10 M NaOH to be between 7.2 and 7.5. Afterwards, the BSA solution (vide supra) was added, yielding a molar ratio of 1:350 (BSA:EDC-FUAc). The mixture was stirred at room temperature for 2 h. Afterwards, the FUAc functionalized BSA (FUAc-BSA) was retrieved by centrifugal filtration (5000  g, 4 8C, 15 min) with Amicon Ultra-15 centrifuge filters (Merck Millipore, Carrigtwohill, Ireland) having a 30 kDa cut-off and washed three times with deionized water. Afterwards, the product was dried by lyophilisation for 24 h yielding a white powder. Yield: 873.3 mg (0.0127  10
3 mol, MW ¼ 68 508). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectra were recorded on an Autoflex Speed by Bruker (Rheinstetten, Germany) in Linear Positive Mode with dihydroxyacetonephosphate (DHAP)-matrix. The binding ratio of FUAc to BSA was determined from the peak shift of FUAc-BSA compared to that of unmodified BSA.

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2.4. Circular Dichroism (CD)-Spectroscopy and UVSpectroscopy CD-spectroscopic analyses were performed with a JASKO J-720 Japan (Tokyo, Japan, settings: band width 1.0 nm, response 1 sec, measurement range 260–183 nm, data pitch 0.1 nm, scanning speed 100 nm min
1, accumulation 10, cell length 0.1 cm, cuvette: quartz). UV-spectroscopic analyses were performed with a JASKO V-550 Japan (Tokyo, Japan, settings: band width 1.0 nm, response slow, measurement range 350–200 nm, data pitch 1 nm, scanning speed 100 nm min
1, cell length 1 cm, cuvette: black quartz, room temperature). BSA and FUAc-BSA were weighted and dissolved in a concentration of 2  10
6 M. Buffer: 10  10
3 M phosphate/ 154  10
3 M NaF, respectively. To guarantee the same protein concentrations for CD-spectroscopy, both proteins were compared by UV-spectroscopy (Figure S1, Supporting Information). The results indicated that FUAc-BSA was in lower concentration in NaF-buffer (1.76  10
6 M) compared to pure BSA. Therefore, CDspectroscopy results were corrected for the comparison of FUAcBSA and BSA. CD-spectroscopy was performed at room temperature, 80 8C and 20 8C (after heating to 80 8C).

2.5. Effects of FUAc and FUAc-BSA on Tumor Cells 2.5.1. Cell Culture The human breast cancer cell lines T-47D and MDA-MB-231 were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% PenStrep in 75 cm2-flasks at 37 8C in the presence of 5% CO2 and passaged when 80–90% of the flask bottom was covered.

2.5.2. Cytostatic Effects of FUAc and FUAc-BSA in MTT-assays The cells were detached from the cell culture flask by trypsinization, counted in a Neubauer cell counting chamber (LO – Laboroptik Ltd, Lancing, United Kingdom) and 5000 cells per well were seeded in 96-well flat bottom plates (BD BioSciences, Heidelberg). After incubation at 37 8C for 24 h, 100 mL of the culture medium were removed from each well and substituted by 200 mL of a 5-FU-, FUAc-, FUAc-BSA-, or BSA-solution in cell culture medium. The initial concentrations of 5-FU and FUAc were adjusted to give 100  10
6 M, 300  10
6 M or 500  10
6 M in the well plates. The concentration of FUAc-BSA solutions was adjusted to give an equivalent concentration of FUAc of 100  10
6 M, 300  10
6 M or 500  10
6 M in the well plates. The equivalent concentration was determined from BSA concentration and the loading parameter (mol FUAc per mol BSA) measured by MALDI-TOF. The concentrations of unmodified BSA were equal to the molar protein concentration in the FUAc-BSA solutions. Each concentration of each substance was measured seven times. Growth control, cells þ 200 mL of RPMI 1640, was seeded six times. On each well plate one well was always used as a blank, 200 mL of RPMI 1640 without cells. The influence of FUAc and FUAc-BSA on the growth of tumor cells was analyzed after 24 h, 48 h, 72 h, and 96 h applying a standard MTT-assay. Briefly, 200 mL medium were removed from

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each well and replaced by 100 mL of sterile MTT-solution (1 mg mL
1 in RPMI 1640). After 2 h at growth conditions the MTT-solution was replaced by 100 mL of a solubilization solution (99.4% DMSO, 0.6% acetic acid and 0.1 g mL
1 SDS) to lyse the tumor cells and solubilize formazan. Then, the plates were agitated for 15 min in the dark and measured at 570 nm in a plate reader (Power Wave 340, Biotek, USA). For each cell line, four measurement series were conducted. For each time (24 h, 48 h, 72 h, and 96 h) and cell line, the mean was calculated. The mean of the growth control was set to 100% and used as a reference value.

(ibidi GmbH, Martinsried, Germany). In each well 15 000 cells were seeded and 200 mL RPMI 1640 medium supplemented with 300  10
6 M BSA/FITC-BSA (9:1)-mixture were added. The cells were examined after 24 h, 48 h, 72 h, and 96 h using an inverted microscope Axiovert 200M equipped with a 100 oil immersion objective (numerical aperture 1.3) and a confocal laser scanning unit Zeiss LSM 510Meta (Zeiss Microimaging GmbH, Jena, Germany). The fluorescence images were obtained by excitation at 488 nm and a long pass emission filter 505 nm. Z-stacks were produced and analyzed applying the LSM 510 software and displayed as overlays of transmission and fluorescence channels in orthogonal section views.

2.5.3. Cytostatic Effects of FUAc and FUAc-BSA in Cell Counting Test For an estimation of the cytostatic effect of 5-FU, FUAc, FUAc-BSA, and pure BSA, cell counting data were collected. For a cell counting test, the tumor cells were detached from the cell culture flask, counted with a Neubauer improved cell counting chamber and seeded with a density of 30 000 cells per well (T-47D: 60 000 cells per well) in a 24-well-plate. After incubation at 37 8C for 24 h, 590 mL of culture medium were removed from each well and substituted by 1180 mL of a 5-FU-, FUAc-, FUAc-BSA-, or BSA-solution in cell culture medium. The initial concentrations of 5-FU and FUAc were adjusted to give 300  10
6 M in the well plates. The concentration of FUAcBSA solutions was adjusted to give an equivalent concentration of FUAc of 300  10
6 M in the well plates. The equivalent concentration was determined from BSA concentration and the loading parameter (mol FUAc per mol BSA) measured by MALDI-TOF. The concentration of unmodified BSA was equal to the molar protein concentration in the FUAc-BSA solutions. Each concentration of each substance was measured three times. Growth control, cells þ 1180 mL of RPMI 1640, was seeded three times. On each well plate, three wells were always used as a blank, 1180 mL of RPMI 1640 without cells. The influence of FUAc and FUAc-BSA on the growth of tumor cells was analyzed after 72 h. To do so, the well’s supernatant (including detached cells) was transferred into 2 mL micro tubes and centrifuged (300  g, room temperature, 10 min). The sedimented cells were re-suspended in 200 mL RPMI 1640 and incubated for 4 min by adding 200 mL of 0.5% trypan blue while gently agitating. Afterwards, the cells were counted with a Neubauer improved cell counting chamber using only the corner squares in the Neubauer cell. In each square the cells were counted with respect to their staining and classified as dead (blue cells), or living (not stained). For counting the attached cells, RPMI 1640 (removed before) was completely replaced by 200 mL Trypsin-EDTA for 13 min at 37 8C to detach the cells from the well’s ground. Then, 200 mL RPMI 1640 were added to inactivate the trypsin. 200 mL of the cell suspension were incubated for 4 min with 200 mL 0.5% trypan blue while gently agitating. Afterwards, the cells were counted as described above. The mean of the growth control was set to 100% and used as reference value. The amount of ‘‘vital cells’’ was calculated by subtracting the number of dead cells from the total number of all cells.

2.6. Confocal Laser Scanning Microscopy (CLSM) The uptake of albumin by the two tumor cell types was investigated by CLSM. The cells were cultured in 8-well m-slides

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2.7. Software For the analysis of data (e.g., calculation of IC50) and its illustration GraphPad Prism 6 was used (San Diego, CA, USA).

3. Results and Discussion 3.1. Functionalization of 5-FU and Binding to BSA The experiments were performed with BSA as a model substance for albumin-carriers in general since BSA and HSA have been found to be highly similar. HSA (585 amino acids) and BSA (583 amino acids) exhibit a sequence homology of amino acids in 76% of the protein.[23–25] It has been shown by spectroscopic analyses that some deviations exist between BSA and HSA in specific regions of the proteins.[24] These distinct differences may influence the cellular uptake in vivo. However, Stehle et al. showed that hepatic uptake of BSA and HSA differed only by 1.1% (8.0% BSA uptake and 6.9% HSA uptake).[26] For a covalent binding of 5-FU to BSA, it is necessary to functionalize 5-FU. The functional unit should allow for binding to free amine groups of a carrier (here BSA) in a peptide fashion, e.g. the free amine group of lysines. Additionally, the side chain bound to 5-FU should be as small as possible to minimize alteration of molecular properties, such as diffusivity. Both criteria are met by an acetic acid side chain that is bound via its methyl group to one of the nitrogen atoms in the 5-FU ring by nucleophilic substitution. The reaction was carried out following a synthesis described by Tada et al.[21] (Scheme 1A). We also verified if a comparable synthesis such as Williamson ether synthesis that has been used in methylcarboxylation of starch[27] is suitable to introduce the acetic acid side chain. We found that obeying a molar ration of 1:1 of chloroacetic acid to 5-FU and heating the reaction mixture to 100 8C for at least 2 h are essential to obtain the mono-methyl-carboxylated 5-FU [2-(5-fluoro-2,4-dioxo3,4-dihydropyrimidine-1(2H)-yl)acetic acid, FUAc]. The successful functionalization was confirmed by 1H-, 19 F- and 13C-NMR. (Spectra of FUAc see Figure S2, S3 and S4).

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Scheme 1. The scheme shows the general procedure of A) FUAc- and B) FUAc-BSA- synthesis (BSA molecular structure adopted from the literature).[48]

For the coupling of FUAc to the free e-amino groups at the end of lysine side chains and an amino-group at the N-terminus of BSA, we used a water-borne carbodiimide based strategy as in linking by active esters (Scheme 1B).[22,28] Major challenges were the water sensitivity of intermediate reaction species that are essential for the proceeding of the coupling of FUAc to BSA. Therefore, controlling pH, optimization of reaction times and stoichiometry were necessary. We found that the most appropriate way to solve these challenges is to work in fairly concentrated protein solutions (BSA 25 mg mL
1) and to apply an exhaustive excess of the acetylating agent FUAc (BSA/FUAc ¼ 1/350 molar ratio). It also improves yield to adjust the amount of EDC and NHS to a stoichiometry of 2 and 3 to FUAc, respectively. The coupling of FUAc to BSA was verified by MALDI-TOF spectrometry. An increase in the molecular peak from 66 306 m/z (BSA) to more than 68 500 m/z (FUAc-BSA) was found (Figure 1A and Supporting Information, Figures S5, S6). On average, this corresponds to approximately 12 FUAc molecules per BSA molecule or to a saturation of 34–40% of all 30–35 accessible lysine binding sites in BSA.[29] Such a loading ratio represents midrange saturation in comparison to other albumin based systems, such as methotrexate-rat serum albumin (MTX-RSA).[17] However, higher degrees of loading have been suspected to drastically change the conformation of albumin and thus yielding to a non-native structure, which leads to accumulation preferentially in the liver and degradation by the reticuloendothelial system.[17,18] Preservation of the

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native conformation of albumin after modification is important for cellular uptake and digestion pathways.[18] To compare the structure of FUAc-BSA and native BSA CDspectroscopy was performed (Figure 1B–D). As it can be seen, both spectra are almost completely overlapping, which confirms clearly that no measurable changes in the secondary structure of BSA occurs after modification to FUAc-BSA. 3.2. Effect of 5-FU, FUAc, and FUAc-BSA on Human Breast Cancer Cells The effect of the conversion of FUAc to 5-FU in terms of its cytostatic potential is currently unclear.[30] Therefore, the cytostatic potential of the FUAc and FUAc-BSA was investigated in comparison to the 5-FU on the human breast cancer cell lines, MDA-MB-231 and T-47D. We applied three concentrations, 100  10
6 M, 300  10
6 M and 500  10
6 M of 5-FU and FUAc. For each reaction, we determined the number of FUAc molecules bound to BSA (NFUAc/BSA) by MALDI-TOF and derived an equivalent concentration for FUAc-BSA (CFUAc-BSA) calculated as follows: C FUAc
BSA ¼ ðC 5
FU Þ  ðN FUAc
BSA Þ
1

ð1Þ

where C5-FU is the applied concentration of 5-FU. In BSA controls, BSA was added at concentrations equal to the corresponding equivalent concentration of FUAc-BSA (Equation 1).

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Figure 1. Characterization of FUAc-BSA (red curve) and BSA (black curve) by means of A) MALDI-TOF- and B–D) CD-spectroscopy. A: For FUAc-BSA the position of the maximal intensity is shifted to approx. 68 500 m/z, with corresponding half-maximum intensities (dotted box) at 67 800 m/z and 69 400 m/z. These values correspond to a binding ratio of 8 and 16, respectively, on average 12 FUAc molecules per BSA molecule. B: BSA and FUAc-BSA dissolved in 10  10
3 M phosphate/154  10
3 M NaF-buffer at room temperature. Blue curve: Difference between BSA and FUAc-BSA calculated by subtraction. C: Similar to B, proteins heated to 80 8C. D: Similar to C, proteins cooled to 20 8C after heating to 80 8C.

3.3. MTT-Assay Cell growth was evaluated by means of MTT-assays after 24 h, 48 h, 72 h, and 96 h (Figure 2). The sensitivity of different cell lines to cytostatic/cytotoxic drugs can be described by the inhibitory concentration 50% (IC50), which is the drug concentration necessary to inhibit 50% of cell proliferation compared to the control. These values for 5-FU, FUAc and FUAc-BSA in our experiments are presented in Table 1. As expected, the strongest inhibition of tumor cell proliferation and the lowest IC50 values were observed for 5-FU that is well known and widely used as a cytostatic drug (Figure 2 and Table 1). While 5-FU showed a strong cytostatic effect after 48 h on both cell lines, for FUAc only a weak proliferation inhibition of MDA-MB-231 was measured at higher concentrations beyond 48 h (Figure 2A1). On T-47D the cytostatic effect was even weaker (Figure 2B1). These changes of cytostatic efficiency can only arise from the chemical modification of the drug. The difference between the chemical structure of FUAc and 5-FU consists in one additional methyl-carboxylic group. As a consequence FUAc is charged under neutral or physiological pH due to the stronger acidity of the carboxyl function (deprotonated at pH > 2) in comparison to the weak acidity of –NH groups in

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the ring (deprotonated at pH >> 8) and it is slightly more hydrophilic than 5-FU. The slight increase in hydrophilicity of FUAc as well as the charge possibly leads to 1) impeded permeation through the hydrophobic zone in the lipid bilayer of the cell membrane and 2) a possible blockade of the cell internalization pathway through uracil carriers that is used by 5-FU. For example, it was found that orotic acid, a C6-carboxylated uracil, cannot use the uracil transporter. Orotic acid crosses cell membranes only by diffusion and, hence, much slower (t1/2: 2890–6930 s) than 5–FU and uracil (t1/2: 25–58 s).[36] A similar mechanism could be valid for FUAc. Additionally, FUAc carries the methyl-carboxylate group on the N1-atom, which is the primary site for any reaction in uridine formation, for instance, fluorodeoxyuridine monophosphate (FdUMP) formation, which is crucial for thymidylate-synthase inhibition, etc. Thus, a weakened or delayed cell proliferation inhibition can be expected from a purely chemical point of view. However, there is still little known on the cellular mechanisms for the cytostatic effects of FUAc, which include, for example, digestion and metabolism of FUAc in cells.[19,30] Interestingly, a strong cytostatic effect on T-47D cells even comparable with that of 5–FU was found for FUAc-BSA (Figure 2B2). For MDA–MB–231 a slightly stronger proliferation inhibition of FUAc-BSA in comparison to FUAc was

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Figure 2. Cells exposed to 5-FU and FUAc (top 1) or to 5-FU and FUAc–BSA (bottom 2) at different concentrations (100  10
6 M, 300  10
6 M, 500  10
6 M) show a time dependent cell growth inhibition. A) Cell line MDA-MB-231, B) Cell line T-47D. The results were obtained from a MTT-assay. Legend: 5-FU 100  10
6 M, 5-FU 300  10
6 M, 5-FU 500  10
6 M, FUAc 100  10
6 M, FUAc 300  10
6 M, FUAc 500  10
6 M, FUAc-BSA 100  10
6 M, FUAc-BSA 300  10
6 M, FUAc-BSA 500  10
6 M.

Table 1. Calculated IC50 values from our MTT-assays compared to those published in literature. The bracketed numbers behind the IC50 value (if quoted) demonstrate the 95% confidence interval and/or additional information.

Substance

5-FU [10


6

M]

Observed (72 h)

Reported

Cell line

Cell line

T-47D

MDA-MB-231

T-47D

MDA-MB-231

Others

50.6

1.32

2.8  0.1 (144 h)[31]

9.3  0.3

–

(37.6–68.1)

(0.07–24.5)

153.75 (48 h, in glucose-

[33]

(72 h)

deprived media)[32] FUAc [10


6

M]

913.5

652

(358–2313)

(502–844)

Not published

Not

0.196 (HeLa, 72 h)[34]

published

and 0.267 (SGC-7901, 72 h)[34] 88.17 (HL-60, 48 h)[35]

FUAc-BSA [10

434


6

M]

88.6

408

(65.6– 19.6)

(238–699)

Not published

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Kinetics and Efficiency of Methyl-Carboxylated 5-Fluorouracil-BSA Adduct www.mbs-journal.de

observed. However, for FUAc-BSA also some recovery appeared at times beyond 48 h. Pure BSA at the equivalent concentrations (Equation 1) did not affect the cell growth significantly (Figure S7, Supporting Information). Thus, the cells that were more resistant to pure FUAc were affected stronger by FUAc-BSA, which is also reflected by the corresponding IC50 values (cell line T-47D, Figure 2 and Table 1). 3.4. Cell Counting In addition to the MTT-assays, cell counting experiments were performed in order to distinguish between cytostatic effect and cytotoxicity. Here, BSA, FUAc, FUAc-BSA, and 5FU were administered at one concentration (300  10
6 M) and for one incubation time (72 h) on the two cell lines MDA-MB-231 (Figure 3A) and T-47D (Figure 3B). Interestingly, nearly no cell death occurred and only 5-FU exhibited a slight cytotoxicity on T-47D cells proving that neither FUAc nor FUAc-BSA are cytotoxic. For FUAc, cell counting showed a stronger cytostatic effect on MDA-MB231 cells (Figure 3A) compared to that found by the MTTassay (Figure 2A1). For T-47D cells the discrepancy occurred even larger with no effect according to MTT (Figure 2B1) and approximately 60% of proliferation inhibition according to cell counting (Figure 3B). This effect seems to be FUAc specific, as we observed the same tendency for other cell lines (e.g., SW-480 and CC-531), which will be subject of a forthcoming publication. We assume that FUAc might interact with the metabolic reduction of MTT and, thus, lead to differing results comparing MTT-assay and cell counting test (see below).

For FUAc-BSA, cell growth of MDA-MB–231 cells is inhibited by 65% and for T-47D by 80%, which is in agreement with the MTT-assay data (Figure 2A2,B2). For pure BSA 300  10
6 M the total number of tumor cells as well as the amount of vital cells is slightly decreased (Figure 3). In the MTT-assay, we found almost no influence of BSA on cell proliferation for MDA-MB-231 (Figure S7A, Supporting Information) and T-47D (Figure S7B, Supporting Information). However, one has to consider the large standard deviations in cell counting and the error of ca. 25% in the MTT-assay of BSA. In general, for vital cells, cell counting yielded smaller cell numbers in comparison to the MTT-assay. For FUAc the MTT-assay results showed deviations by factor of 2.2 to 3.8 compared to cell counting data. This strong deviation may arise from analysis specific errors. The MTT-assay is a spectroscopic analysis of the 1-(4,5-dimethylthiazol-2-yl)3,5-diphenylformazan concentration, a reduction product of MTT. The reaction is catalyzed by cell metabolic enzymes, nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) or other easy oxidizable groups such as the thiol group in cystein.[37,38] Thus, the formazan concentration gives within some error direct access to cells’ metabolic activity. It is assumed that cell activity can be directly related to the number of vital cells in a sample, which is only true for very specific and well defined conditions (e.g., same metabolic activity).[39] Therefore, an altered metabolism of the tumor cells due to FUAc might influence the accuracy of the MTT-test. According to Luo et al. and Chung et al. an intracellular conversion of FUAc to 5-FU seems less probable.[19,30] Acetate is activated by acetyl-CoA synthetase leading to

Figure 3. A) Cell counts in percentage relative to the controls after 72 h: MDA-MB-231 cells and B) T-47D cells when BSA, FUAc, FUAc-BSA, and 5-FU were added to RPMI 1640 with a concentration of 300  10
6 M (equivalent concentration related to 5-FU). Legend: total number vital cells, dead cells. of cells,

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Table 2. Tumor cell specific protein expression.

Protein expression Cell line T-47D

[44,47]

MDA-MB-231

[14,44]

SPARC

mABP

–

þ

–

–

acetyl-coenzyme A (acetyl-CoA).[40] Amongst other, acetylCoA is essential for the synthesis of fatty acids and for energy generation by entering the citric-acid cycle.[41,42] One completed citric-acid cycle produces three molecules of NADH which are responsible for the reduction of MTT to formazan (as mentioned above). In fact, Liu describes the acceleration of citrate oxidation due to increased availability of acetyl-CoA for prostate cancer cells as a

Figure 4. CLSM. For better understanding, the upper panel schematically demonstrates the development of orthogonal CLSM-section view. The orange, waved arrows represent the microscope’s optical pathway. A) The yellow ball within the cube is sliced through the dotted x-yplane. The projection is on top of the cube. B) Identical to (A), with projection on x-z-plane. C) Identical to (A), with projection on y-z-plane. D) All projections are summarized in the cube. The x-z-plane and the y-z-plane are unfolded to one level with x-y-plane. E) Final orthogonal CLSM-image. The lower panel shows confocal laser scanning micrographs of tumor cells after 72 h incubation with 300  10
6 M BSA/FITC-BSA (ratio 9/1) in orthogonal section view and overlay of transmission and fluorescence mode: left: MDA-MB-231, right: T-47D.

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source of energy confirming this hypothesis.[43] The increase in NADH production by the citric-acid cycle could be an explanation of the strong deviation between MTTassay and cell counting for the FUAc samples. MTT-assay results indicate an increased metabolic activity due to higher levels of NADH. Cell counting assays, however, quantify the cell number directly, which is not influenced by the increased metabolic activity. Thus, in the MTT-assays FUAc specific overestimation of cell vitality is observed. To understand the effect of FUAc-BSA the possible uptake and metabolic pathways have to be discussed. For the interaction of albumin with cells, two pathways are currently under consideration: the SPARC (secreted protein, acidic and rich in cysteine) pathway, where the cell secrets a protein into the extracellular matrix that binds albumin and leads to endocytosis, and the mABP (plasmamembrane albumin-binding protein) pathway. The SPARC pathway has recently been under heavy investigation due to improvements in nab-Paclitaxel application and the related cellular response.[14] The mABP pathway was studied in recent experiments due to the proliferation inhibition of estrogen sensitive cells, such as MCF-7 or T-47D by albumin.[44] This inhibitory property of albumin has been shown to be more pronounced for HSA and less pronounced for BSA.[45] However, this inhibition was found for concentration variations in the range of 2.5–10% FBS.[46] In our experiments FBS concentration was 10% and the amount of BSA added to the cell culture was negligible (0.5–0.275%). The cell lines MDA-MB-231 and T-47D investigated in this study differ in their receptor expression pattern in general (Table S3, Supporting Information) and in mABP-protein expression pattern in particular (Table 2), Hence, one could expect differences in the quantity of internalized albumin by these cells.

strong fluorescence signal appears heterogeneously distributed in vesicle like structures inside the cells. Such a distribution is characteristic for an uptake via endocytosis. Enrichment of fluorescence is also found in the confocal images of MDA-MB-231 cells (Figure 4 lower panel, left). However, the internalized FITC-BSA is obviously less than in T-47D cells. Additionally, the increased fluorescence is limited to regions close to the cell membranes or inbetween the cells forming the cluster. Almost no fluorescence can be detected deeper in the cytoplasm as visualized in the orthogonal section view (Figure 4, lower panel left). These observations suggest that cellular uptake of BSA is in fact less effective in MDA-MB-231 cells than in T-47D cells, in agreement with the specific expression of mABP (Table 2). Taking this into account the results obtained by the MTT-assay and cell counting for FUAc–BSA can be interpreted as follows: MDA-MB–231 cells take up FUAcBSA in amounts, where digestion initially leads to cell proliferation inhibition but only to an extent that can be compensated by the cell, e.g., through energy gain by BSA digestion. T-47D cells take up larger amounts of FUAc-BSA, that strongly inhibits cell proliferation. Luo et al. described the hypothesis that most probably 5FU is released when FUAc is coupled to a macromolecule.[30] Obviously, the question arises why the cell lines handle the FUAc-BSA uptake differently. We reason that this difference is associated with unspecific uptake by pinocytosis for MDA-MB-231 (weak overall uptake) and effective mABP driven endocytosis and pinocytosis for T-47D (strong overall uptake). As a result T-47D cells are affected stronger by FUAc-BSA, which yields after digestion most-likely 5-FU[19,30], than MDA-MB-231 cells although MDA-MB-231 cells are more sensitive to FUAc.

4. Conclusion 3.5. CLSM In order to estimate the uptake of BSA, the two tumor cell lines were cultured in the presence of FITC-BSA and investigated using the z-stack option of CLSM. Figure 4 shows representative orthogonal section views of z-stacks of tumor cells after 72 h incubation with a 300  10
6 M BSA/FITC-BSA (9:1)-mixture as overlays of transmission and fluorescence modes. The right red framed box displays the y-z-plane of the cut along the vertical red line and the upper green framed box represents the x-z-plane of the cut along the horizontal green line. The blue line in the x-z and y-z plane indicates the z-position of the x-y-plane shown in the central image. A cluster of adherently growing T-47D cells can be seen in Figure 4 lower panel, right. The overlay of the images in transmission and fluorescence mode allows for recognition of the FITC-BSA distribution in the cells. It can be seen that a

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The clinically established uracil antimetabolite 5-FU was successfully modified by methyl-carboxylation to FUAc and coupled to BSA in an average ratio of 12 mol FUAc per mol BSA without changing the structure of the protein. The cytostatic effects of FUAc and FUAc-BSA were investigated in vitro by means of MTT-assays and cellcounting-tests. Both human breast cancer cell lines, MDAMB-231 and T-47D were highly susceptible to 5-FU. When adding FUAc overestimation of cell vitality in MTT-assays compared to cell counting was found in both cell lines, which could be explained assuming an intracellular conversion from FUAc to 5-FU and acetate combined with resulting activation of the citric-acid cycle. We highly recommend utilizing a second assay when a new drug is investigated in vitro. The cytostatic effect of FUAC-BSA on both cell lines was shown in different extent and with some deviations by both

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applied assays. This is the most promising result of the present study. Our results suggest that not only the influence of the well characterized cancer cell protein SPARC is responsible for the endocytotic uptake of human serum albumin-/BSA-bound (e.g., Nab-Paclitaxel) or covalently coupled drugs like our FUAc-BSA, but also mABP may play an important role. FUAc-BSA will have undoubtedly many clinical advantages in comparison to 5-FU, such as reduced adverse effects due to specific uptake by tumor cells (e.g. myelosuppression) and similar effectiveness. Further evaluation is necessary, but testing the tumorous expression of SPARC and mABP could be crucial for successful treatment of patients with albumin-coupled drugs.

Acknowledgements: CD- and UV-spectroscopic studies were performed at the Leibniz-Institute of Molecular Pharmacology (Working Group: Peptide-Lipid Interaction – Peptide Transport). The authors thank Heike Nikolenko for her excellent technical assistance. Human mammary cancer cell line T-47D was kindly provided by Prof. Kurt Possinger (Department of Hematology €tsmedizin Berlin, Germany). and Oncology, Charit
e – Universita Human mammary cancer cell line MDA–MB–231 was kindly € ftner (Department of Hematology and provided by PD Dr. Diana Lu €tsmedizin Berlin, Germany). The Oncology, Charit
e – Universita authors are also thankful for 1H-NMR-spectroscopy which was performed by the working group ‘‘NMR’’ (Head: Prof. Dr. Clemens € gge), Institute of Chemistry, Humboldt-University Berlin. Mu Funding Sources: Federal Ministry of Economy and Technology of Germany (BMWi) ZIM- KF2354402FR0, Charit
e-Research Grant 89508160. Results presented here are part of the doctoral thesis of M.J.K. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. Received: August 5, 2013; Revised: September 27, 2013; Published online: November 8, 2013; DOI: 10.1002/mabi.201300363 Keywords: albumin; drug delivery systems; 5-fluorouracil; human breast cancer cell lines; MALDI

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