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Unveiling the Self-assembling Behavior of 5-Fluorouracil and its N,N'dimethyl Derivative : A Spectroscopic and Microscopic Approach Pavel Banerjee, Devdeep Mukherjee, Tapas Kumar Maiti, and Nilmoni Sarkar Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02378 • Publication Date (Web): 20 Sep 2017 Downloaded from http://pubs.acs.org on September 24, 2017
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Unveiling the Self-assembling Behavior of 5-Fluorouracil and its N,N'-dimethyl Derivative : A Spectroscopic and Microscopic Approach
Pavel Banerjee1, Devdeep Mukherjee2, Tapas Kumar Maiti2 and Nilmoni Sarkar*1 1
Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India
2
Department of Biotechnology, Indian Institute of Technology Kharagpur, India
*Corresponding Author: Nilmoni Sarkar E-mail:
[email protected] Fax: 91-3222-255303
Abstract At physiological conditions, 5-fluorouracil (5-FU), an anti-cancer drug self-assembles into fibrils by strong hydrogen bonding network while its methylated derivative, 5-fluoro-1,3-dimethyluracil (5-FDMU) do not make fibril due to lack of strong hydrogen bonding motif. In vitro, 5-FU selfassembly is sensitive to physicochemical conditions like the pH and ionic strength of the solution, which tune the strength of the non-covalent driving forces. Here we report a surprising finding that the buffer-which is necessary to control the pH and is typically considered to be inert-also significantly, influences 5-FU self-assembly which indicates an important role of counter-ions in the fibril formation. We have also monitored concentration and time dependent fibrillar growth of 5-FU. Again, fibril growth process is probed in dynamic condition using microfluidic platform. The self assembly of 5-FU compared to its methylated derivative, shows lower cytotoxicity to the cultured human erythroleukemic cells (K562 cells), which plausibly states the reason behind the more effectiveness of 5-FU derivative drugs than 5-FU itself.
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1. Introduction. Nature utilizes molecular self-assembly as an efficient tool to design advanced functional structures from simple building blocks like amino acids, peptides, nucleic acids, and phospholipids.1,2 A number of degenerative disorders including Alzheimer’s disease,3 Parkinson’s disease,4 and spongiform encephalopathy5 are related with the formation of amyloid fibrils by peptides and proteins. Recently, pioneering work by Gazit and co-workers have showed how even an unprotected single amino acid (phenylalanine), from evaporation of a solution on a surface, can assemble to well-ordered amyloid like fibrils.6 Later, it is reported that tyrosine, glycine also form fibrils in neutral aqueous solution.7,8 It is also reported that the fibrils formed by single amino acids are the major cause of the neurodegenerative disorders like phenylketonuria (PKU) and tyrosinemia type II.6,8 As most of the metabolic disorders are rare, a confined amount of targeted research has been conducted so far. However, the study about the formation of fibrils by small molecules gains an increasing attention nowadays. Gazit and coworkers have reported recently that like single amino acids, single nucleobases like adenine, uracil also can self-assemble in a fibrillar fashion.9 5-fluorouracil (5-FU), remains the backbone of modern chemotherapy, is widely used as an anticancer drug after first development near about 60 years ago by Heidelberg et al. 10 After that, 5-FU is extensively studied in photophysical11,12 and medical field13 to develop new strategies to increase the anticancer activity of 5-FU and for better understanding of the mechanism of action. However, drug resistance remains a significant concern to the clinical use of 5-FU.13 Due to this reason, 5-FU is used in higher doses, sometimes in millimolar (mM) concentrations.14,15 Due to the use of high concentration of 5-FU, many side effects are generated including cardiotoxicity,16 and hepatotoxicity.17 Inside the cell,5-FU is converted to 5-fluorodeoxyuridine monophosphate (5-FdUMP). 5-FdUMP competes with deoxyuridine (dUMP) for thymidylate synthase (TS). TS is an enzyme which involves in the conversion of dUMP to deoxythymidine monophosphate (dTMP). Due to this involvement of 5-FU with TS, the formation of dTMP is interrupted and thus DNA synthesis is stopped.13 5-FU contains both strong hydrogen bond donor (i.e. –NH) and acceptor (i.e. –CO) groups which guide its self assembly process. Hulme et al. have showed that 5-FU forms ribbon like morphology by making two pairs of hydrogen bonds between –NH and –CO.18Sun et al. have reported fibril formation of 5-FU dilysine conjugates.19Significant role of hydrogen bonding on 2 Environment ACS Paragon Plus
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the aggregation behavior of small organic molecules is reported in literature.20,21On the other hand, N-methylation at the 1- and 3- positions of 5-FU makes 5-fluoro-1,3-dimethyluracil (5FDMU) which lacks strong hydrogen bond donors.22,23 The stabilizing forces are weak CH···O hydrogen bonds and dipole-induced dipole interactions in 5-FDMU.22 Recently, Majhi et al. have shown the effect of methylation on the self-assembly of 4-aminophthalimide which indicates a prominent role of hydrogen bonding on the self-assembly of small molecules.24There are several reports in literature which suggest that N-methylated derivatives of 5-FU (like capecitabine, gemcitabine etc.) are more advantageous with fewer side-effects in cancer treatment
25,26
but no
strong reason is reported behind it till date. To the best of our knowledge, this is the first report explaining the reason (i.e, fibril formation) behind the lower effectiveness of 5-FU than its derivatives. Inspired by the single nucleobase fibril formation 9 and the difference between the degree of selfassembling forces present in 5-FU and 5-FDMU
18,22
, we have explored the ability of 5-FU and
5-FDMU to form different kind of aggregates under mM concentrations. In this article, we have showed that 5-FU but not 5-FDMU, can form fibrils in phosphate buffer (PBS, 0.1 M, pH 7.4) by using a series of microscopic and spectroscopic tools. We have also monitored the fibril forming behavior of 5-FU in different types of buffers to have a flavour of self assembling mechanism of 5-FU. Concentration and time dependent fibril formation of 5-FU are also examined. Microfluidics is employed to examine the growth of 5-FU fibrils in dynamic flow condition. Finally, the effect of fibril formation of 5-FU on the viability of K562 cells is also studied. Thus, the obtained result will improve our understanding of the mechanism of showing the more effectiveness of 5-FU derivative drugs than 5-FU itself and the microfluidic technique provides the real time images of 5-FU fibrils. 2. Experimental Section. 2.1. Materials. 5-Fluorouracil (5-FU) and 5-Fluoro-1,3-dimethyluracil (5-FDMU) were purchased from Sigma Aldrich. 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4Hpyran (DCM) dye was used as received from Exciton. Anhydrous sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic (NaH2PO4), sodium acetate (CH3COONa), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), tris buffer, bicine buffer were obtained from SRL (India). Acetic acid (CH3COOH) was acquired from Merck. All the chemicals were used without further purification. The buffer solutions are prepared following the conventional 3 Environment ACS Paragon Plus
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methods. Human blood was collected from two healthy male donors attending Medical College, Kolkata for regular health check-up. A written consent was taken from the subjects. This study was performed according to the guidelines of Indian Council of Medical Research, IIT Kharagpur and the Helsinki Declaration. The blood samples were allowed to clot at room temperature for 45 min. Then the samples were centrifuged at 2000 x g for 10 min at 40C. The supernatant was then collected carefully and stored at -200C until further use. Throughout the experiments mili-Q water (ddH2O) was used. The resistivity of the mili-Q water is 18.2 MΩ·cm at 25 °C in our set up. The chemical structures of all the chemicals are shown in Scheme 1.
Scheme 1. Chemical Structures of 5-FU, 5-FDMU and DCM. 2.2. Preparation of the Fibrillar Samples. 5-FU and 5-FDMU are dissolved in different buffers (i.e. PBS (pH ~ 7.4), acetate buffer (pH ~ 5.6), bicarbonate buffer (pH ~ 9.2), tris-HCl buffer (pH ~ 7.4), bicine buffer (pH ~ 7.4), all buffer concentrations are 0.1M) and double distilled H2O to prepare 5 mg/ml (~38 mM) concentration. We have kept the solution for 24 h at room temperature for the preparation of fibrillar assemblies. All experiments are done in that mentioned concentration (5 mg/ml). 2.3. DCM Fluorescence study. The formation of 5-FU fibrils was initially monitored by measuring the fluorescence intensity of DCM. The concentrations of fibrils and DCM were maintained at 5mg/ml and 10 µM, respectively. The fluorescence intensity of DCM was recorded in both neat PBS and in PBS fibrillar solution to confirm fibril formation using Spex Fluorolog-3 (model FL3-11) spectrofluorimeter. Again, the study in different buffers have continued
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following same manner. The samples were excited at 480 nm. The slit width of the monochromator is 2/1 (excitation/emission) for all measurements. 2.4. Maintenance of cell lines. K562 (Chronic Myeloid Leukemia) Cell line was obtained from National Center for Cell Science, India and maintained inside humidified 5% CO2 incubator with regular subculture in Roswell Park Memorial Institute medium (RPMI-1640).
Media was
supplemented with 10% Fetal Bovine Serum (FBS), 100 units of Penicillin, and 0.1 mg Streptomycin. 2.5. Cell viability assay. K562 cells were suspended in serum free RPMI medium and seeded in a 96-well plate at a density of 1× 104 cells /well. Cells were then incubated with the indicated concentrations of 5-FU and 5-FDMU (each measurement for three different concentrations, i.e., 2.5, 5 and 7.5 mg/ml) for 6h. Then, MTT assay was performed as described elsewhere.27The absorbance was measured at 570 nm using a microplate reader (Thermo). 2.6. Microfluidic experiments. Microfluidic channels were made with poly (dimethylsiloxane) (PDMS). A silicone master was fabricated by contact photolithography using SU8 as described elsewhere.28A mixture of PDMS and cross-linker (10:1 w/w) was poured onto the mold, degassed, heated at 65 oC for 2 h. Finally, the PDMS channels were gently taken out and inletoutlet ports were punched. Subsequently the channels were treated with oxygen plasma and gently press-bonded against glass slides. 5-FU solution was passed through the channels by a syringe pump (Harvard Apparatus, USA). The growths of the fibrils were monitored using a Fluorescent Microscope (Oympus). Captured images were analyzed using ImageJ 1.45S. 2.7. Instrumentation. The different microscopic pictures of 5-FU and 5-FDMU in different buffers are visualized by electron microscopy (FESEM, HR-TEM) and fluorescence lifetime imaging microscopy (FLIM) techniques. The comparative studies of fibrillar rigidity are performed by DCM fluorescence and fluorescence correlation spectroscopy (FCS) measurement. The involvement of different bonds in fibril formation are studied by fourier transform infrared spectroscopy (FTIR). The detailed description of the instruments is discussed in the supporting information. The details of the fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS) are given below. 2.7.1. Fluorescence Lifetime Imaging Microscopy (FLIM) and Fluorescence Correlation Spectroscopy (FCS). FLIM and FCS were used to characterize the formation of 5-FU fibrils and 5 Environment ACS Paragon Plus
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5-FDMU aggregates. Both the experiments were carried out using DCS 120 confocal laser scanning FLIM system (Becker &Hickl DCS-120) equipped with an inverted optical microscope of Zeiss with a 20X (FLIM) and 40X(FCS) objective. DCM is used as a fluorescent marker in both FLIM and FCS techniques. The sample was excited using a 488 nm diode laser, working in the pulse mode (10 mW, a repetition rate of 50 MHz), and a long pass filter (498 nm) was used to separate the fluorescence signal from the excitation source. The fluorescence lifetime was measured by a polarized dual-channel confocal scanning instrument (Becker & Hickl DCS-120) attached to an output port of the microscope. It was controlled by a galvo-drive unit (Becker & Hickl GDA- 120). For the lifetime images, a polarizing beam splitter and two independent HMP100-40 GaAsP hybrid detectors (ID-Quantique ID100) were equipped with DCS-120. The polarized fluorescence transient obtained by time-correlated single photon counting detection electronics (Becker & Hickl SPC- 152, PHD-400-N reference diode) was used to generate the images. The instrument response function of the system is less than 100 ps fwhm (full width at half-maximum). About 10 µL of the sample solution is placed on a slide and allowed to be dried before taking the images. In each case, images were taken at 15 min interval and they exhibited the same position for the individual solutions, which confirmed proper immobilization of samples on the glass surface, and they remained stationary during data acquisition. For all the FCS experiments, the final concentration of probe molecules (DCM) was 2 nM and for FILM it was 6 µM. FCS correlates the fluorescence fluctuations which originate from the variation in the concentration of the fluorescent species due to the translational motion or the chemical reaction or complex formation into or out of the confocal volume (~1 femtolitre). The fluorescence autocorrelation function can be defined as =
(1)
In the above equation, is the fluorescence fluctuation at time t, + is the fluctuation after a delay and is the average fluorescence intensity. The details of the instrumentation are described in our earlier publication.29 A single-component diffusion equation has been used in order to fit the autocorrelation functions.
= 1 +
1 +
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(2)
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In this equation, N is the average number of fluorescent molecules in the detection volume, and is the average time of fluorescent molecules diffusing in the detection volume with their corresponding amplitude (A). S denotes the structure parameter of the excitation volume and it is defined as (l/r), where l is the longitudinal radii and r is the transverse radii. Transverse radii (r) can be determined through the fitting of an autocorrelation curve of a fluorescent species with known diffusion coefficient. We have used R6G in water for this purpose and the diffusion coefficient is 426µm2s-1
30
and the autocorrelation curve of R6G in water (spectra not
shown) is fitted with the following equation in order to determine the global parameter r and S.
= 1 +
1 +
!.#
(3)
Here, is the diffusion coefficient of the fluorescent species. In the fitting analysis, r and S are kept as linked global parameter. S=5 is obtained after fitting the correlation curves of R6G in water and observation volume ($%&& is obtained from the following equation, $%&& = ' (/* + ( ,
(4)
The final value of + is obtained as 365 nm and $%&& is 1.35 fl. All the FCS experiments were performed at room temperature and the diffusion coefficient can be obtained from the following equation,
=
(5)
3. Results and Discussion. 3.1. Monitoring the difference of Aggregates formed by 5-FU and 5-FDMU. The aim of this article is to investigate the different self-assembling pattern of 5-FU and 5FDMU. Hence, initially the evidence for the formation of fibrils has been provided. Firstly, we have investigated the formation of 5-FU and 5-FDMU aggregates by using DCM (4(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran) fluorescence as used by Zhang and co-workers.31 Our group has also reported earlier that DCM can efficiently indicate the fibril formation of single amino acids.32,33 In this experiment, 5-FU aggregates show significance enhancement in fluorescence intensity while 5-FDMU shows very little enhancement (Figure S1). The formation of different type of aggregates is further investigated by
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FLIM technique for 5-FU and 5-FDMU. It is observed that 5-FU self-assembles to form amyloid like fibrillar structures with a persistence length in the order of a few micrometers (µm) at 5mg/ml concentration [Figure 1-(a)-(i),(ii)] while 5-FDMU forms smaller discrete aggregates (not like fibril) at the same concentration [Figure 1-(a)-(v),(vi)]. To confirm the structures, which are observed for dried samples, also present in the solution, we have taken FLIM in solution also [Figure 1-(a)-(iii), (iv)]. In solution, we also have observed 5-FU fibrils at the same concentration (i.e. 38 mM). [Figure 1-(a)-(ii), (iv), (vi)] are the intensity images calculated from the photons in all time channels of the pixels. In the FLIM image, the lifetime obtained for each pixel is enclosed by colour [Figure 1-(a)-(i), (iii), (v)], and the lifetime distribution of the entire image is shown in [Figure 1-(b)-(i), (ii)]. We have also taken the FLIM images of 6 µM DCM in PBS and in tris buffer as blank experiment (Figure S5) where no aggregation is found. This confirms that the aggregations found in Figure 1- (a) are solely due to 5-FU and 5-FDMU self-assembly.
(a)
(b)
Figure 1. (a) Fluorescence lifetime and intensity images of 5-FU fibrils after solvent evaporation [(i), (ii)], 5-FU fibrils in solution phase [(iii), (iv)] and 5-FDMU aggregates [(v), (vi)] (scale bars are 1.05 µm, 1.6 µm and 2.01 µm respectively). (b) Lifetime distributions of DCM in (i) 5-FU fibrils and (ii) 5-FDMU aggregates obtained from the FLIM images. FLIM images provide information about the lifetime distribution of the dye within different kind of aggregates. As the fluorescence lifetime of the dye is sensitive to the surrounding environment, 8 Environment ACS Paragon Plus
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one can easily predict the environmental rigidity from lifetime distribution analysis. The dye molecules are in a physically inhomogeneous environment for the heterogeneity of the aggregates formed. The width of the lifetime distribution depends on the heterogeneity parameter.34 It is very difficult to obtain a particular lifetime value from the FLIM images as the lifetime values vary in different regions of the aggregates.35,36 The broader lifetime distribution also implies that DCM faces different heterogeneous milieu in different regions of formed aggregates37 which are confirmed by FLIM and FESEM techniques. The lifetime distributions of DCM inside 5-FU and 5-FDMU aggregates are shown in [Figure 1-(b)-(i),(ii)]. DCM exhibits a lifetime distribution in between 1700-2750 ps with a maximum at around 2400 ps in 5-FU fibrils whereas in 5-FDMU aggregates, it shows a distribution in between 1700-2291 ps with a maximum around 2075 ps. In our previous study,32 it is observed that in lifetime distribution, a distinct peak is generated on the lower lifetime side when inhibition of fibril occurs, i.e, surroundings become less compact. Therefore, it can be stated that 5-FU affords a better hydrophobic environment to DCM than 5FDMU aggregates. For further confirmation, we have employed field emission scanning electron microscopy (FESEM) and high-resolution transmission electron microscopy (HR-TEM) to observe both the morphologies. These images are well correlated with the FLIM images [Figure 2- (a)- (i)- (iv)].
(a) (b)
Figure 2. (a) FESEM and HR-TEM images of 5-FU fibrils[(i),(ii)] and 5-FDMU aggregates[(iii),(iv)] (scale bars for FESEM images are 3 µm and 500 µm, for HR-TEM 9 Environment ACS Paragon Plus
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images scale bars are 1 µm and 200 nm respectively). (b) FCS traces of DCM in only PBS (blank, black), 5-FU fibrils (red) and 5-FDMU aggregates (blue). Therefore, these microscopic techniques confirm the fibril formation of 5-FU and also no fibril formation of 5-FDMU. In addition, the autocorrelation traces of DCM in both the aggregates are also examined comparatively with the help of FCS measurement. FCS utilizes fluctuations in fluorescent signals arises due to some dynamic processes, mainly translational diffusion into and out of observed volume. Given its ability to detect species that differ in size over several orders of magnitude, FCS is an ideal technique to identify and track the ensemble of species that form during amyloid assembly/disassembly over time.38-40The fitted traces are represented in [Figure 2-(b)]. All FCS traces are fitted by a single component diffusion model (as mentioned earlier). After fitting, the diffusion time of the probe molecules are determined which are used to calculate the values of diffusion coefficient in each case. The diffusion coefficients of the fluorophore in these systems are calculated from an average of three data sets. It is reported that the diffusion coefficient (Dt) of DCM in neat water is 300 µm2 s-1.41 We also get almost same value in neat PBS (~302 µm2 s-1). Upon binding with 5-FU fibrils, Dt value becomes 1.36 times slower (~220 µm2 s-1) compared to neat PBS while in 5-FDMU aggregates, Dt value retards to ~281 µm2 s-1. Recently, Kim et al.42 and Sarkar and co-workers43 have measured the number density of the fluorescing species from FCS to get an idea of the fluorescence came from aggregate structures of molecule. In our case, we have also compared the number density of the fluorescing species in both 5-FU fibrils and 5-FDMU aggregates (Figure S2). It is observed that the number density of the fluorescent species in the diffusing volume has increased from 5-FDMU aggregates to 5-FU fibrils. The presence of less number of fluorescing species in 5-FDMU aggregates as compared to 5-FU fibrils suggests that the aggregation is more compact in case of 5-FU fibrils. Sasmal et al. also have suggested that as a highly hydrophobic probe, DCM prefers to be confined to the hydrophobic part of the aggregation.41 Therefore, as a hydrophobic probe, when DCM enters into large and compact fibrils of 5-FU, significant retardation in diffusion coefficient has been observed compared to neat PBS and 5-FDMU aggregates. To investigate the molecular level information about the fibril formation, fourier transform infrared spectroscopy (FTIR) is employed. The FTIR spectra of 5-FU and 5-FDMU in PBS (both aggregated and non-aggregated form) are depicted in (Figure S3). The stretching vibrations of -C=O of 5-FU appear at 1723 cm1
(for -C2=O) and 1663 cm-1 (for -C4=O) when it presents in non-fibrillar form.44 We have also
got similar stretching frequency of carbonyl for 5-FU in water where no fibril is formed [Figure 10 Environment ACS Paragon Plus
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S3 – (b)]. However, after fibril formation, there occurs only a single peak for -C=O groups of 5FU at 1690 cm-1 which indicates that two carbonyl groups become equivalent in nature. For –NH region (3100-3500 cm-1),44 there also a broadening occurs for fibrillar solution of 5-FU which further supports the presence of two identical –NH groups. For 5-FDMU, there is no fibril formation and hence we have obtained almost no shift of carbonyl peaks before and after smaller aggregates formation. Therefore, the above discussion dictates the involvement of -C=O and – NH group for fibril formation of 5-FU. Different research groups have used density functional theory (DFT) studies18,45 and X-ray crystal data.22,46,47,48 to analyze the driving force behind the self-assembly of 5-FU and 5-FDMU. Recently, Mohamed et al. have shown that the interactions behind the dimer formation of 5-FU in aqueous solution by DFT studies.49 All these reports support our microscopic and spectroscopic observations of different kind of aggregates formed by 5-FU and 5-FDMU. As mentioned earlier, 5-FDMU doesn’t have any –NH group and hence cannot form strong hydrogen bond network as 5-FU to form fibril.18,22 Some smaller aggregates are formed due to CH···O interaction which is very weak compare to -NH···OC- hydrogen bonding.22 3.2. Concentration and Time dependent Fibril Formation of 5-FU. As 5-FU is an anti-cancer drug, we are interested to study the concentration as well as time dependent study of fibril formation in PBS. Several groups have reported concentration dependent study of single amino acids (L-phenylalanine, L-tyrosine, glycine).7,8 Perween et al. have found that at µM solution there is no fibril formation (some small spherical aggregates) of L-Phenylalanine but at mM solution, there is dense fibrils.7 We have also monitored fibril formation of 5-FU from 1 mM to 38 mM (our experimental concentration) concentration [Figure 3-(a)-(i)-(vi)]. It is observed that at 1 mM concentration [Figure 3-(a)-(i)], there is no significant aggregates. At 5 mM concentration [Figure 3-(a)-(ii)], a spherical morphology consisting of rod like aggregates is found. A single rod type aggregate is observed at 8 mM concentration [Figure 3-(a)-(iii)]. Fibrils are started to grow from 10 mM concentration [Figure 3-(a)-(iv)] and at 15 mM concentration there are significant fibrils [Figure 3-(a)-(v)]. Figure [3-(a)-(vi)] shows 5-FU fibrils at 38 mM concentration (i.e. our working condition). All these images are taken after 24h of sample preparation.
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(b)
Figure 3. Fluorescence lifetime images of 5-FU fibrils in PBS (a) Concentration dependent(i) 1 mM (scale bar is 6.8 µm), (ii) 5 mM (scale bar is 1.75 µm), (iii) 8 mM (scale bar is 2.5 µm), (iv) 10 mM (scale bar is 1.75 µm), (v) 15 mM (scale bar is 8.05 µm), (vi) 38 mM (scale bar is 1.75 µm) (all images are taken after 24h of sample preparation) (b) Time dependent(i) 2h (scale bar is 7.05 µm), (ii) 4h (scale bar is 2.65 µm), (iii) 6h (scale bar is 16.15µm), (iv) 8h (scale bar is 3.25µm), (v) 12h (scale bar is 8.8µm), (vi) 16h (scale bar is 5.5 µm) (all images are taken for 38 mM 5-FU fibrils). We have also monitored fibrillar growth of 38 mM 5-FU fibril with respect to time [Figure 3-(b)] .Recently Gazit and co-workers have studied time dependent growth of Boc-FF fibrils and they found a change in morphology from spherical to nanotube like structure.50 Bianco and coworkers have also studied time dependent fibril formation of L-tyrosine.8 We have also observed similar type of morphological change at around 8h [Figure 3-(b)-(iv)].The different color of FLIM images arises due to the different lifetimes of DCM for the heterogeneity of the aggregates35-37as discussed earlier. So, it can be summarised that 5-FU fibrils started to grow at ~10 mM concentration and a 38 mM 5-FU fibril takes ~8h to initiate growing. 3.3. Roles of pH, ionic strength and counter-ion of buffers in fibril formation of 5-FU.
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(a)
(b)
Figure 4. (a) Fluorescence lifetime and intensity images of 5-FU fibrils/aggregates in acetate buffer [(i), (ii); scale bar is 2.1 µm], bicarbonate buffer [(iii), (iv); scale bar is 3.78 µm], tris buffer [(v), (vi); scale bar is 1.65 µm], bicine buffer [(vii), (viii); scale bar is 2.4 µm],water [(ix), (x); scale bar is 9.69 µm],human serum [(xi), (xii); scale bar is 1.05 µm]. (b) FCS traces of DCM in 5-FU fibrils/aggregates in different buffers. To study the sensitivity of the 5-FU self-assembly to physicochemical conditions like the solution pH40,51and ionic strength 52-54 , which tune the strength of the non-covalent driving force, we have proceed further to study 5-FU self-assembly in different conditions. We have observed a surprising finding that the buffer-which is necessary to control the pH and is typically considered to be inert-also significantly, influences 5-FU self-assembly. Recently, several groups have 13 Environment ACS Paragon Plus
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reported buffer mediated effect on self-assembling of fibrillar system.55,56 In anionic buffers (i.e., acetate, bicarbonate, PBS), it is noticed that fibrils have helical structures but in cationic (i.e., tris) and zwitterionic (i.e, bicine) buffers, fibrils are flower shaped (Figure 4). We have also shown the fibril formation of 5-FU in human serum, a more physiologically relevant environment [Figure 4-(a)-(xi), (xii)]. All the above FLIM observations are confirmed by FESEM technique (Figure 5).
Figure 5. FESEM images of 5-FU fibrils/aggregates in (a)acetate buffer(scale bar is 10 µm) (b)bicarbonate buffer(scale bar is 20 µm) (c)tris buffer(scale bar is 10 µm) (d)bicine buffer(scale bar is 100 µm) (e)water(scale bar is 500 µm). Hulme et al. hypothesized that in presence of water, the hydrogen bonding network of 5-FU is disturbed due to tight solvation of -CO and -NH functional groups by water molecules.18 Moreover, from literature report, it is evident that depending on the environment, 5-FU can selfassemble into different structures and shapes.57,58Qiu et al have reported that in presence of cation, uracil can form quintet like structure (alike flower).59 The aggregation of doxorubicin in different buffers was attributed to the bridge effect of the multianion by Zhu et al.56 In our case, we also believe that the ions play the same role of joining the neighbouring 5-FU molecules, since no aggregate was found when 5-FU was dissolved in water. The microscopic results also suggest that counter-ion increases compactness of 5-FU fibril and this is supported from the work of Bowers and co-workers.60 To find a comparative picture about the rigidity of fibrils formed by 5-FU in different buffers, we have taken the help of DCM fluorescence and FCS technique. In DCM fluorescence, it is obtained that more prominent enhancement in fluorescence 14 Environment ACS Paragon Plus
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intensity in the case of three anionic buffers while for the cationic and zwitterionic buffers, the enhancements are small (Figure S4). In water, 5-FU shows almost no enhancement in fluorescence intensity. These give an indication about the extent of 5-FU fibril formation in different types of buffer. In the FCS measurement, we have obtained different Dt values for 5-FU fibrils in different buffers by fitting all the autocorrelation traces using a single component diffusion model which are shown in [Figure 4-(b)] and tabulated in (Table 1). Table 1 : The diffusion parameters obtained by fitting the fluorescence correlation curves of DCM in 5-FU Fibrils and 5-FDMU aggregates formed in different buffers.
Compound
5-FU
5-FDMU
BLANK (only PBS)
Buffer
-(Sec)
Dt (µm2 Sec-1)
Acetate
227±11.0
146±7.0
0.97
Bicarbonate 152±8.0
219±11.0
0.96
PBS
151±8.0
220±12.0
0.96
Tris
127±7.0
262±15.0
0.96
Bicine
126±7.0
264±15.0
0.96
Water
112±6.0
297±15.0
0.97
PBS
118±8.0
281±20.0
0.94
PBS
110±7.0
301±20.0
R2
0.95
The results corroborate our microscopic and steady state observations. As no significant aggregate forms in water, Dt value remains almost same (~ 296 µm2 s-1) as in neat PBS. For anionic buffers, Dt values retard most because larger and compact helical like morphologies are formed whereas for cationic and zwitterionic buffers, the retardation of Dt values are in between 15 Environment ACS Paragon Plus
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the Dt values for anionic buffers and 5-FDMU aggregates which indicate that a different type of fibrillar morphology is formed. Comparing between the buffers used at pH ~7.4 (i.e. PBS, tris, bicine), it can be stated that they are completely different in nature (i.e. anionic, cationic, zwitterionic respectively). It is reported that zwitterionic buffer compounds are indeed expected to have little effect on the ionic strength of a solution.52 It is also reported that PBS gives 7 times more ions than a zwitterionic buffer in solution.53,54 So, the ionic strength and different counterions of these pH~7.4 buffers are the governing factors for different types of morphology and hydrophobic environment of 5-FU assemblies. In the case of three anionic buffers (i.e. PBS, acetate, bicarbonate), we have observed that for acetate buffer, the fibrils are formed in the largest extent. To explain this, we have studied a pH dependent fibril formation study of 5-FU in three different pH (5.6, 7.4, 8.0) of PBS. From DCM fluorescence (Figure S6), FLIM [Figure 6(a)] and FCS [Figure 6-(b)], we have found that the extent of fibril formation is comparable in case of pH~7.4 and pH~8.0 (in PBS) which is a similar observation of comparing the fibril formation in PBS and bicarbonate buffer. At pH~5.6 (in PBS), the extent of fibril formation is the largest among the three different pH of PBS we have taken. It suggests that at acidic pH, fibrils are formed to a larger extent. For acetate buffer, both the effects of pH and counter-ions are present simultaneously, which make it the most favourable condition for the self-assembly of 5FU.
(a)
(b)
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Figure 6. (a) Fluorescence lifetime images of 5-FU fibrils at different pH of PBS (i) pH~5.6 (scale bar is 4.25 µm), (ii) pH~7.4 (scale bar is 1.75 µm), (iii) pH~8.0 (scale bar is 2.75 µm). (b) FCS traces of DCM in 5-FU fibrils at different pH of PBS.
3.4. Microfluidic study to Monitor 5-FU Fibrillar Growth in Dynamic Flow Condition.
(a)
(b)
Figure 7. (a) Microfluidic set up (b) Elongation of 5-FU fibril (30 min interval between images, scale bar is 20µm). In recent times, microfluidic channels have been used to monitor a single nanofiber in real time.61-63Such system has a unique advantage of providing in vivo-like micro-confinement and to monitor the fibril growth profile in dynamic condition. In addition, high surface area to volume ratio in microfluidic channel often results in augmented reaction rate. Here, we have examined growth of 5-FU fibril in fluid flow condition. Initially, freshly prepared 5-FU solution (5mg/ml in acetate buffer) was injected into a straight microfluidic channel having 50 µm height, 20 mm length and 1.2 mm width at a flow rate of 1µl/min. The flow rate was optimised following
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previously published reports.61,63 We could visualise the fibres after ~ 4 h of continuous fluid flow at the mentioned flow rate. The formation and elongation behaviour of the needle-like structures were examined under light microscope [Figure 7-(b)].We have observed approximately 15µm elongation of the fibril in 2.5 h. This suggests that the apart from buffer composition, formation of 5-FU fibril also critically depends on the fluid dynamics. We observed all fibre growths adjacent to the wall and elongation towards the direction of the fluid flow.64All experiments in the microfluidic channel have been monitored thrice to obtain precise growth rate. A movie frame is also made by assembling the collected images (time gap 25s) (Movie S1).It is very helpful to visualize the motion of fibrils in dynamic condition inside the microfluidic channel. 3.5. Cell Viability Assay.
Figure 8. K562 cell viability studies using MTT assay after 6 hours exposure to (a) 5-FU fibrillar solution (b) 5-FU initial solution (i.e. just after adding 5-FU in PBS, no fibrils formed) (c) 5-FU fibrillar solution after centrifugation (i.e. fibrils are destroyed) (d) 5-FU solution in water (e) 5-FDMU aggregate solution. (For each case, viabilities are measured in three different concentrations).
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As it is observed that 5-FU assembles under physiological conditions, the cell viability of 5-FU becomes an attractive issue. We have investigated cytotoxicity of 5-FU in three different experimental conditions: i) just after addition of 5-FU to PBS (initial time), ii) after fibril formation by 5-FU, iii) after centrifugation of the 5-FU fibrillar solution. For each condition, cell viabilities are measured for three different concentrations using 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) assay which indicate a dose dependent cytotoxic effect on the cells [Figure 8- (a), (b), (c)]. As 5-FU is itself an anti-cancer drug, it is expected to show lower cell viability in all conditions. But surprisingly, for the fibrillar solution of 5-FU [Figure 8(a)], even for the highest concentration, the cell viability is more than what obtained from its nonfibrillar conditions [Figure 8-(b), (c)]. In initial condition (i.e., no fibril is formed), 5-FU shows higher cytotoxicity even at the lowest concentration. Again when the fibrillar solution is centrifuged (i.e., the fibrillar morphology is destroyed),9 cell viability decreases drastically. We have extended our cell viability measurement to the 5-FU solution in water [Figure 8-(d)] (also no fibril is formed) and got similar type of low cell viability as non-fibrillar solutions in PBS. Cell viability of 5-FDMU aggregates solution is also studied where it shows lower values in all three concentrations (
