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The supervisors of this project: Dr. María Blanca Ros Latienda (Full professor of the University of Zaragoza) and Dr. Teresa Sierra Travieso (Scientific Researcher of the CSIC), of the Institute of Materials Science of Aragon (University of Zaragoza-CSIC), CERTIFY That the present work called “Thermosensitive and photopolymerizable hydrogels based on Pluronic® F127” has been carried out by I. Lucía Herrer Jiménez under our supervision. This document reflects the work done as the Final Master Project to obtain the Master Degree in Nanostructured Materials for Nanotechnology Applications of the University of Zaragoza. Zaragoza, 9th September 2013 Dr. María Blanca Ros Latienda Dr. Teresa Sierra Travieso ABSTRACT Here it is reported the design, synthesis and characterization of new Pluronic® F127 derivatives, with the ability to form thermosensitive and photopolymerizable hydrogels. These active hydrogels undergo a sol-to-gel transition by increasing the temperature in a physiologically important temperature range thus resulting attractive for biomedical applications and drug delivery systems. Pluronic® F127 has been functionalized with photoreactive groups and the obtained derivatives and their precursors have been fully characterized by conventional techniques. Then the aimed compounds have been processed as macroscopically molded hydrogels and as nanostructured hydrogels (nanogels). A highly crosslinked internal structure has been reached by the photopolymerization technique for the thermosensitive macroscopic hydrogels designed to act as cell scaffolds for cartilage repair. Swelling and degradation studies as well as their morphological characterization by SEM have been carried out. Concerning the nanostructured hydrogels (nanogels), after determining its critical micellar concentration and applying a photopolymerization process to fix the nanostructure, they have been characterized by TEM, SEM and DLS. Cell viability assays have been carried out for both types of system, the macroscopic hydrogel and the nanogel. Acronyms CGC Critical gelling concentration CMC Critical micelle concentration CMT Critical micelle temperature DLS Dynamic light scattering DCS Differential scanning calorimetry DCM Dichloromethane DMEM Dulbecco‟s Modified Eagle‟s Medium GPC Gel permeation chromatography IHs Injectable hydrogels IR Infrared LCST Lower critical solution temperature MALDI Matrix-Assisted Laser Desorption/Ionization MS Mass spectrometry MSC Mesenquimal stem cells NMR-1H Proton nuclear magnetic resonance NMR-13C Carbon nuclear magnetic resonance PBS Phosphate buffered saline SEM Scanning electron microscopy TEM Transmission electron microscopy TIM Test-tube inverting method UV Ultraviolet INDEX 1. INTRODUCTION ........................................................................................................ 1 2. OBJECTIVE ................................................................................................................. 3 3. PLANNING .................................................................................................................. 4 4. RESULTS AND DISCUSSSION ................................................................................. 6 4.1. SYNTHESIS AND CHARACTERIZATION ....................................................... 6 4.1.1. Synthesis of the linear Pluronic derivative, P/A-2. ....................................... 6 4.1.2. Synthesis of the hybrid dendritic-linear-dendritic block copolymer P/A-4. ………………………………………………………………………………….11 4.2. SOL-GEL STUDIES............................................................................................ 12 4.2.1. Sol-Gel properties of commercial Pluronic F127 .................................... 14 4.2.2. Sol-Gel properties of P/C-2 ...................................................................... 15 4.2.3. Sol-Gel properties of P/A-2...................................................................... 16 4.3. PHOTOPOLYMERIZED HYDROGELS ........................................................... 17 4.3.1. MACROSCOPIC HYDROGEL ........................................................................ 17 4.3.1.1.-Photopolymerized hydrogel ................................................................ 17 4.3.1.2.-Morphological characterization .......................................................... 18 4.3.1.3.-Swelling and degradation .................................................................... 20 4.3.2. NANOGEL (NANOSTRUCTURED HYDROGEL) ............................................. 22 4.3.2.1.-Critical micelle concentration (CMC) determination ......................... 22 4.3.2.2.-Photopolymerized nanogel.................................................................. 23 4.3.2.3.-Morphological characterization .......................................................... 24 4.4. CELL VIABILITY ASSAYS .............................................................................. 27 4.4.1.MACROSCOPIC HYDROGEL ......................................................................... 27 4.4.1.1.-Results of 2D cell viability assays ...................................................... 29 4.4.1.2.-Result s of 3D cell viability asssays .................................................... 31 4.4.1.3.-No photopolymerized vs. photopolymerized hydrogel. Material P/A-2. ............................................................................................................................. 31 4.4.2.NANOGEL .................................................................................................... 33 5. EXPERIMENTAL SECTION .................................................................................... 34 5.1. Synthesis of the linear pluronic derivative ........................................................... 34 5.2. Synthesis of the hybrid dendritic-linear-dendritic block copolymer.................... 38 5.3. TIM and DSC procedure ...................................................................................... 43 5.4. Photopolymerized macroscopic hydrogel obtention ............................................ 43 5.5. Swelling and degradation procedure .................................................................... 44 5.6. Photopolymerized nanogel obtention. .................................................................. 45 5.7. MSCs and HeLa cell culture and sample preparation. ......................................... 46 6.-CONCLUSIONS ........................................................................................................ 47 7.-REFERENCES ........................................................................................................... 47 Appendix 1 ......................................................................................................................... I Appendix 2 .................................................................................................................... VII 7 In the 1H-NMR spectrum of the P/NP-2 compound (spectrum 2 in Figure 1), three new characteristic signals appear with respect to P. First, the multiplet at 4.41 ppm which corresponds to PEG chain protons directly linked to the newly formed carbonate group (G protons). The other two characteristic signals are at 7.39 ppm (J protons) and 8.27 ppm (K protons) corresponding to the aromatic protons. These aromatic signals help to follow the next reaction due to its complete disappearance in the spectrum of the compound P/C-2 (spectrum 3 in Figure 1). Finally, in the last spectrum (spectrum 4 in Figure 1), corresponding to the P/A-2 molecule, two significant changes are appreciated: three doublets of doublets between 5.84 and 6.44 ppm, which are the signals of the introduced acrylate groups (M, L and M‟), and the multiplet at 4.22 ppm corresponding now to G protons and J protons. OOO 67 HO 99 H 99 A B C D E OOOOOO 67 98 98 O O A B C D E F G HN H OH H N HO I J O2NO O OOOOO 67 98 98 O NO2 O O A B CD E F G H I J K L OOO 67 O 98 98 A B C D E OOH N O O F G H I JO O N H O O K L M,M' 1 2 3 4 Figure 1. 1H-NMR spectra of commercial Pluronic F127 (P), P/NP-2, P/C-2, and P/A-2, respectivelly. 8 The molecule P/C-2 is difficult to characterize only by 1H-NMR because meaningful protons are mobile protons, then, another useful tool to check the changes within the molecule was infrared spectroscopy (IR), as shown in Figure 2. In the IR spectrum of the P/C-2 compound (red spectrum in Figure 2), it can be appreciate a band between 3500-3250 cm-1 corresponding to the O-H st, which help to confirm the structure of the molecule. Also, another important signal which can be compared between the molecules, is the band appearing at 1765 cm-1 for the P/NP-2 compound (blue spectrum in figure 2), corresponding to the C=O st from carbonate, and at 1722 cm-1 and at 1728 cm-1 for the compound P/C-2 and P/A-4, respectively, corresponding to the C=O st from carbamate. Also, mass spectrometry (MS) has been used to study the compounds P/C-2 and P/A-2 to prove the mass changes with respect to the original material, P. Figure 2. FTIR-ATR spectra of P, P/C-2 and P/A-2 respectively. 9 In Figure 3, a displacement of the mass distribution towards higher mass/charge relationship is observed, which is due to the functionalization carried out at the initial polymer, P. Finally, both compounds were characterized by Gel Permeation Chromatography (GPC), using polystyrene standards for calibration. (see Figure 4) 4944,8 13106,2 0 50 100 150 200 250 300 350 400 05000 10000 15000 20000 25000 30000 Intensity (%) m/z 5936,3 13608,6 0 100 200 300 400 500 600 700 05000 10000 15000 20000 25000 30000 Intensity (%) m/z 5938,6 13644 0 100 200 300 400 500 600 700 800 05000 10000 15000 20000 25000 30000 Intensity (%) m/z Figure 3. MS of P and its derivatives. P P/C-2 P/A-2 a) b) Figure 4. Obtained chromatograms by GPC for a) P/C-2 compound and b) P/A-2 compound. 10 The obtained data are presented in Table 1. Mn a Mw PDI b MWc calculated (g/mol) Pluronic F127 16748 17193 1.03 12600 6438 7105 1.1 P/C-2 13898 14621 1.05 12774 P/A-2 13445 14159 1.05 12882 Commercial Pluronic F127 shows a chromatogram with two different molecular weight distributions (see Appendix 1). The new materials show also two distributions in their chromatograms, but they are not so well separated, this behavior could be associated to the new functionalization present in the molecules. The new materials show two distributions in their chromatograms corresponding to two molecular weight distributions as shown by the commercial Pluronic F127 (see Appendix 1). Also, as indicated in Table 1, comparing the obtained values from GPC referring the calculated ones, MW (last column of Table 1), we find differences. These differences can be attributed, in one side to the fact that calibration is made with a different polymer, polystyrene, which has a different hydrodynamic volume. On the other side, different variables to take into account are how the polymer behaves within the column, and the polymer‟s molecular weight. The use of this technique also allows us to verify the presence of two molecular weight distributions in both materials, as previously noted by MS. Table 1. Obtained data from GPC experiments performed with Styrage® columns HR1. aMn : number average molecular weight (g/mol) obtained by GPC. bPDI: polidispersity index (Mw/Mn) obtained by GPC. cMW calculated: molecular weight in g/mol, calculated as the sum of the molecular weight of individual blocks. 11 4.1.2) Synthesis of the hybrid dendritic-linear-dendritic block copolymer (HDLDBC), P/A-4. The synthetic route followed to prepare the compound P/A-4 is shown in Scheme 3: HO OOOH 99 67 99 O O OOO 67 99 O O 99 H O OO H OOO 67 99 O 99 O O OH OH HO HO O O OOO 67 99 O O 99 O OO O O O O O OO O NO2 NO2 O2N O2N O O OOO 67 99 O 99 O ONH O O O N H N H O N H OH HO HO OH O O O OOO 67 99 O 99 O O H N O O O O N H H NO N H O O OO O O O O O O O O OO O O H H DMAP dry DCM H2 Pd/Ccat. AcOEt Cl O O NO2 dry Py dry DCM H2NOH dry DCM O Cl TEA dry DCM O P P/B-2 P/OH-4 P/NP-4 P/C-4 P/A-4 Scheme 3. Synthetic route of compound P/A-4. 12 The products P/B-2, P/OH-4, P/NP-4, and P/C-4 have been correctly obtained and isolated, but, the optimal experimental conditions to obtain the final product, P/A-4 have not been stated so far. After using different strategies, the full functionalization of the ending hydroxyl groups of P/C-4, was not reached. In all attemps, a certain degree of functionalization, but not constant, was observed. The best results, even though functionalization was not complete, were achieved using tetrahydrofuran as solvent instead of dichloromethane. The optimization of this reaction is still in progress. 4.2.-SOL-GEL STUDIES The sol-gel properties have been studied for the following materials: -Commercial Pluronic F127. -Linear Pluronic derivative, P/A-2 and its precursor, P/C-2. For each material, aqueous solutions with five different concentrations (w/v) have been tested, i.e. 16 %, 18 %, 20 %, 23 %, and 25 %. (Experimental section 5.3) The study of these properties is based in the application of two different methods to determine the Low Critical Solution Temperature, (LCST):  Test-tube inverting method (TIM): This simple method was employed to roughly determine the sol-gel transition temperature. When the test-tube, or vial, containing the solution, is inverted, it is defined as a sol phase if the solution deforms by flow, or a gel phase if there is no flow. Accordingly, the LCST was defined as the temperature when the material doesn‟t flow during at least 10 seconds when the vial is upside down. Also, in this experiment the materials were heated above the LCST, to find the temperature at which the material became again a sol, after reaching the gel state, what has been called gel-sol transition. 13  Differential Scanning Calorimetry (DSC): DSC is a thermoanalytic technique that allows determining sol-gel transition temperatures, and which has been already used for the study of Pluronic F127 16, 17. In our case, for each studied material, the same samples prepared for the TIM, have been studied by DSC technique. The obtained thermograms allow the recognition of two different signals, corresponding to the micellization and gelation processes (Figure 5). The main peak corresponds to the micellization process. It allows to know the critical micellization temperature (CMT), at which the aggregation of the polymeric chains, for a particular concentration takes place. As it may be appreciated in Figure 5, the gelation peak appears as a small peak at higher temperature, this peak is only well defined when the polymer concentration is high, usually 25 %(w/v)18, for smaller concentrations this peak is not so well defined. In the cases where the gelation peak was not clearly appreciated in the thermogram, but the material showed sol-gel properties, we stated as the value for LCST as the temperature at which it is considered that the gel is entirely formed, corresponding this temperature with the stabilization of the baseline in the thermogram ( Figure 6). -1,5 -1 -0,5 0 0,5 1 1,5 2 2,5 3 010 20 30 Derivative heat flow (mW/min) Temperature (°C) Figure 5. Example of obtained thermograms from P/A-2 material, illustrating the micellization and gelation processes. P/A-2 25 %(w/v) Micellization Sol-gel transition 14 The obtained DSC thermograms for all the studied materials are presented in Appendix 2. 4.2.1 .- Sol-Gel properties of commercial Pluronic F127 Figure 7 shows the phase diagram of this material, which shows how Pluronic F127 behaves depending on concentration and temperature. Table 2 summarizes the sol-gel properties obtained from the application of the two above-commented methods, the test-tube inversion method (TIM) and the differential scanning calorimetry (DSC). 15 25 35 45 55 65 75 85 15 17 19 21 23 25 27 Temperature (°C) Concentration % (w/v) P Phase Diagram Sol Sol Gel Figure 7. Phase diagram for Pluronic F127, obtained from TIM. P/C-2 23 %(w/v) Figure 6. DSC thermogram obtained for the material P/C-2 at 23 % (w/v). Example to illustrate how the LSCT is determined for materials where the gelation peak is not detected. 15 Micellization Temp. (°C) LCST (gelation process)(°C) Gel-sol transition (°C) % [P] (Obtained by DSC) TIM DCS (baseline)* DCS (gel peak) TIM 16,5 14 37 32 - 59 18 13 36 30 - 65 20 11 26 25 - 68 23 10 21 26 18 76 25 9 18 30 17 78 4.2.2 .- Sol-Gel properties of P/C-2 Results concerning the gelling properties for P/C-2 are shown in Figure 8 and Table 3. LCST (gelation process)(ᵒC) Gel-sol transition (ᵒC) % [P/C-2] Micellization T. (ᵒC) TIM DCS (baseline)* TIM 16 13 no gel no gel - 18 13 no gel no gel - 20 11 36 29 48 23 10 26 25 62 25 9 22 25 70 15 25 35 45 55 65 75 15 17 19 21 23 25 27 Temperature (°C) Concentration % (w/v) P/C-2 Phase diagram Sol Sol Gel Table 2. Summary of the obtained data for Pluronic F127 with TIM and DSC methods. Table 3. Summary of the obtained data for P/C-2 with TIM and DSC methods. Figure 8. Phase diagram for P/C-2 , obtained from TIM. *Data obtained as illustrated in Figure 6. *Data obtained as illustrated in Figure 6. 16 Compound P/C-2, which has hydroxyl groups as terminal groups in both parts of the molecule, shows a different behaviour in terms of gelation properties compared to Pluronic F127. Indeed, it is necessary, due to the chemical modification within the molecule, to increment the concentration of the compound up to 20% (w/v) to obtain a hydrogel. 4.2.3.- Sol-Gel properties of P/A-2 Finally, results concerning the gelling properties for P/A-2, are shown in Figure 9 and Table 4. Compounds P/C-2 and P/A-2 behave similarly with respect to the critical gelling concentration (CGC), 20 % (w/v), which is not affected by the fact of changing the size of the side groups of the molecule. However, for P/A-2 the temperature range for the gel state is bigger than for compound P/C-2. This leads us to conclude that the presence of acrylate groups allows the stabilization of the gel. 15 25 35 45 55 65 75 15 17 19 21 23 25 27 T emperature (° C) Concentration % (w/v) P/A-2 Phase Diagram Sol Sol Gel Figure 9. Phase diagram for P/A-2, obtained from TIM . 23 From where we obtain the values of CMCP/C-2 = 0.034 %(w/v), and CMC P/A-2 = 0.011 %(w/v), by establishing the intersection of the straights. These CMC are much lower than the one of the starting material, Pluronic F127, which has a CMC = 0.25 % (w/v)20. This significant (one order of magnitude) decrease of the CMC could minimize or overcome the problem that occurs with Pluronic, for which dissociation of the micelles upon dilution leads to the release of encapsulated drug before it reaches the targeted site. Another important fact to underline is the lower CMC for the P/A-2 material with respect to P/C-2 material. This result proves that modifying the ending groups from hydroxyl to acrylate groups, micelle formation becomes easier, requiring lower concentration for micelles to form. 4.3.2.2.-Photopolymerized nanogels Nanogels were prepared by disolving P/A-2 in distilled water containing the photoinitiator Irgacure 2959 (see Experimental section 5.6). Then, the solution was UVirradiated and next, the photopolymerized nanogels were dyalized during 24 hours and finally they were filtered23. The correct photopolymerization of the nanogels was checked by 1H NMR, observing that all signals corresponding to unreacted acrylate groups disappear after the photopolymerization process (Figure 16). a) b) Figure 16. 1H-NMR of P/A-2 material. a) original synthesized compound. b) Photopolymerized nanogel, dialyzed, filtered, lyophilized, redissolved in distilled water, lyophilized again and solved in CDCl3. 24 The photopolymerized nanogels obtained in this way, were named “PDF nanogels”, what means they have been photopolymerized, dialyzed and filtered. The characteristics of these PDF nanogels where checked by SEM, TEM and DLS. Another performed test on these nanogels was to submit the PDF nanogel to a lyophilization process, getting a solid material. This solid was mixed with distilled water to verify that its properties were maintained. Checking the characteristic of this redissolved nanogel also by TEM, SEM and DLS. The nanogels obtained in this way are named “PDF re-dis nanogels”, what means they are photopolymerized, dialyzed, filtered, lyophilized and re-dissolved again. The aim of this experiment was twofold: One, to know if the material withstands manipulations for future applications in which the fact of having a solid material could represent some advantages. Second to know that micelles don‟t become an indissoluble aggregate block. 4.3.2.3.-Morphological characterization To characterize the morphology of the photopolymerized P/A-2 nanogel, TEM and SEM have been used. Throught both techniques it has been proven the formation of nanostructures which can be interpreted as aggregates of micelles. The rounded shape is present for the PDF nanogel and also for the PDF re-dis nanogel, as can be perfectly appreciate in the SEM images (Figures 17, 18, 19 and 20).  Characterization by TEM - P/A-2 PDF nanogel Figure 17a. TEM images of photopolymerized, dialyzed and filtered P/A-2 nanogel. 25 - P/A-2 PDF re-dis nanogel  Characterization by SEM - P/A-2 PDF nanogel Figure 17b. TEM images of photopolymerized, dialyzed and filtered P/A-2 nanogel. Figure 18. TEM images of photopolymerized, dialyzed, filtered, lyophilized and redissolved P/A-2 nanogel. Figure 19. SEM images of photopolymerized, dialyzed and filtered P/A-2 nanogel. 26 - P/A-2 PDF re-dis nanogel Also, thanks to this characterization, we can confirm that the micellar aggregates don‟t aggregate further, once the material is lyophilized and subsequently dissolved again in distilled water.  Size distributions Results concerning the size distribution of the observed nanostructures are summarized in Table 5. P/A-2 PDF nanogel diameter P/A-2 PDF re-dissolved nanogel diameter SEM 52.5 -172.8 nm 42.460.8 nm TEM (average) * 56.9 nm 135.8 nm DLS Effective diameter 52.0 ±1.2 nm 83.8 ±1.0 Mean diameter 74.9 nm 143.9 nm In the case of P/A-2 PDF nanogel, the diameter obtained with different techniques results coherent, because the TEM value is within the range of SEM values, and the mean diameter obtained by DLS, is also a reasonable value, taking into account that DLS technique provides the hydrodynamic diameter. Figure 20. SEM images of photopolymerized, dialyzed and filtered P/A-2 nanogel. Table 5. Summary of sizes obtained from SEM, TEM and DLS, for the P/A-2 nanogel. (* average value calculated from more than 10 structures.) 27 In contrast, more divergent data are obtained in the case of P/A-2 re-dissolved nanogel. These variations can be attributed to a possible further aggregation between nanoaggregates but deeper studies should be carried out. 4.4.-CELL VIABILITY TESTS Taking into account the potential biomedical applications of these materials, cell viability assays are needed. Cell viability assays were carried out for both macroscopic hydrogels and for nanogels with two different cells types: - Human mesenchymal stem cells (MSCs), study carried out on the macroscopic hydrogels in collaboration with Dr. María José Martinez, from Blood and Tissue Bank of Aragon. - HeLa cells, study carried out on the nanogels in collaboration with Dr. Pilar Martín-Duque, from Araid Foundation and Rebeca González, from IIS Aragón, Biomedical Research Center of Aragon. Concerning MSCs, the two main characteristics of these cells are24,25: - Adherence to plastic. MSC must be plastic-adherent when maintained in standard culture conditions using tissue culture flasks. - Multipotent differentiation potential. MSCs are able to differentiate into a number of mesenchymal phenotypes, including those that form bone, cartilage, muscle, fat and other connective tissues. Concerning HeLa cells, they are a human epithelial adenocarcinoma cell line. This cell line is the oldest and most widely used human cell line26. 4.4.1.MACROSCOPIC HYDROGEL In order to check the cell viability of P, P/C-2 and P/A-2 when forming macroscopic hydrogels, tests were performed with cells MSCs. 28 2D and 3D experiments (Figure 21) have been performed to evaluate cell viability in the hydrogels prepared. 2D cell culture was performed by covering the cells layer with the hydrogel, while, in 3D cell culture, the hydrogel was formed in presence of the cells, then cells were distributed within the hydrogel. Cells growth and sample preparation are described in the Experimental section 5.7. Cell viability assays have been performed for: - Pluronic F127, and its hydrogels, in order to compare it with our derivatives. - Compound P/C-2, and its hydrogels. - Compound P/A-2, and its non photopolymerized and photopolymerized hydrogels. The assessment of cell vialibility was performed with the LIVE/DEAD® Viability/Cytotoxicity Kit, which can discriminate live from dead cells by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity27, as shown in Figure 22. (For further information, see Appendix 2.) Figure 21. Illustration of 2D and 3D cell culture 3 D Figure 22. Example of images taken from the fluorescence inverse microscope of stained cells with the LIVE/DEAD® KIT. Images of a)Pluronic F127, 2D, 24h, 20 %(w/v).b)P/C-2, 2D,24h, 20 %(w/v).c)P/A-2, 2D, 24h, 20 %(w/v). d)Pluronic F127, 2D, 48h, 20% wt, dead cells. 2D 3D 29 Cell viability for the three materials was checked at 24, 48 and 72 hours by using a fluorescence inverted microscope. All experiments were performed in triplicate. In each of the three wells for each experiment, two images were taken from different areas of the well, therefore the results shown, are an average of six values. The percentage of living cells was determined by counting living cells and dead cells, and referring to cell viability as the percentage of the amount of living cells over total present cells. All control samples, containing only cells and DMEM, presented a cell viability above the 98%. The chosen concentrations to carry out the cell viability assays, have been the same for all tested materials: - 5 %(w/v), 10 %(w/v). At these concentrations, none of the materials are forming the hydrogel, therefore it is considered that the monomers are in solution. - 20 %(w/v), 23 %(w/v). Concentrations where the thermoresponsive hydrogel is formed in all materials. 4.4.1.1.-Results of 2D cell viability assays. The results obtained from the 2D cell viability assays, for Pluronic F127, P/C-2, P/A-2 and their hydrogels, are presented in Figure 23. Figure 23 shows the variations on the cell viability depending on the material, its concentration, and time. Take notice that for these assays, the material P/A-2 was not photopolymerized. 30 From these comparative studies different conclusions can be obtained: - All materials in solution, cause a decrease in cell viability when increasing the concentration from 5 to 10%, Figure 23 a) and b). This fact is not so clearly observed when the material is forming the physical gel, comparing Figure 23 c) and d), even for some cases, the viability is higher for the higher concentration. - In general terms, except for the material P/A-2 at 23 %, in the gel state, Figure 23 c) and d), our materials have a higher cell viability than the original Pluronic F127, showing in some cases up to 40% difference. - The best cell viability is obtained for the material P/C-2. 0 20 40 60 80 100 24 48 72 Cell viability % Time (h) Studied materials in solution, 5 % (w/v). Pluronic, 5% P/C-2, 5% P/A-2, 5% 0 20 40 60 80 100 24 48 72 Cell viability % Time (h) Studied materials in solution, 10 % (w/v). Pluronic, 10% P/C-2,10% P/A-2,10% 0 20 40 60 80 100 24 48 72 Cell viability % Time (h) Studied materials, 2D tests at 20 % (w/v). Pluronic 2D 20 % P/C-2 2D 20% P/A-2,2D,20% 0 20 40 60 80 100 24 48 72 Cell viability % Time (h) Studied materials, 2D tests at 23 % (w/v). Pluronic, 2D, 23% P/C-2, 2D, 23% P/A-2, 2D, 23% a) b) c) d) Figure 23. Results from 2D cell viability assays at different concentrations. a)5 % (w/v), b) 10 %(w/v), c) 20 % (w/v), d)23 %(w/v). 31 4.4.1.2.-Results of 3D cell viability assays. Figure 24 shows the results obtained from the cell viability assays carried out with the same materials in a 3D system, where cells are distributed within the hydrogel network. For these assays the material P/A-2 was not photopolymerized. In this case it can not be drawn a clear conclusion about the relationship between cell vibility and concentration. What can be appreciated again, is that the best cell viability corresponds to the samples based on the material P/C-2 for both concentrations, and the worst one corresponds for those based on the acrylated material P/A-2. Nevertheless, during measurements, it was observed for the 3D experiments that the staining providing the fluorescent green color, was much more difficult to get, and consequently, is was more difficult to quantify the alive cells in these experiments. This difficulty has been associated to a lower diffusion rate, due to the fact of working with a dye kit not originally designed for the assays in gel samples. 4.4.1.3.-Non-photopolymerized vs. photopolymerized hydrogel. Material P/A-2. In order to compare the difference between the material based on a physical network or on a physical-chemical network, the results obtained for the P/A-2 material at different concentrations, non-photopolymerized and photopolymerized, are presented in Figure 25. 0 20 40 60 80 100 24 48 72 Cell viability % Time (h) Studied material, 3D tests at 20 % (w/v). Pluronic, 3D, 20% P/C-2, 3D, 20% P/A-2, 3D, 20% 0 20 40 60 80 100 24 48 72 Cell viability % Time (h) Studied material, 3D tests at 23 % (w/v). Pluronic, 3D, 23% P/C-2, 3D, 23% P/A-2, 3D, 23% Figure 24. Results from 3D cell viability assays at different concentrations. e) 20 % (w/v), f) 23 %(w/v). e) f) 32 Comparing the 2D assays with 3D assays, a clear decrease in the cell viability can be appreciated concerning both concentrations and the dregree of crosslinking within the hydrogels. We have tentatively attributed these results to a limited diffusion within the polymeric network. In almost all cases, when the material is photopolymerized, the cell viability decreases, regardless the concentration and the test type. One possibility, to explain this result is the drowning of the cells due to the internal structure fixed by the photopolymerization process, providing an 0 10 20 30 40 50 60 70 80 90 100 24 48 72 Cell viability % Time (h) 2D P/A-2 2D 20% no photopolymerized 2D 20% photopolymerized 2D 23% no photopolymerized 2D 23% photopolymerized 0 10 20 30 40 50 60 70 80 90 100 24 48 72 Cell viability % Time (h) 3D P/A-2 3D 20% no photopolymerized 3D 20% photopolymerized 3D 23% no photopolymerized 3D 23% photopolymerized Figure 25. Results obtained from cell viability assays. Comparison between P/A-2 material, photopolymerized and no photopolymerized, at different concentrationsin 2D and 3D assays. g) h) 39 1H-RMN (300 MHz, CDCl3): δ (ppm): 1.11 (s, 3H, G), 3.70 (d, J=11.7 Hz, 2H, F), 4.64 (d, J=11.4 Hz, 2H, F´), 5.49 (s, 1H, E), 7.37 (m, 3H, A, B), 7.47 (m, 2H, C). 13C-RMN (75 MHz, CDCl3): δ (ppm): 17.7 (G), 42.1 (H), 73.4 (F), 101.9 (E), 126.2 (C), 128.3 (B), 129.0 (A), 137.5 (D), 178.8 (I). IR (cm-1, KBr): 3005 (OH), 2865 (C-H st), 1702 (C=O). -Synthesis of Benzylidene-2,2-bis(oxymethyl)propionic anhydride. In a flask, benzylidene-2,2-bis(oxymethyl)propionic acid, (11.0 g, 49.6 mmol) and DCC (5.7 g, 27.7 mmol) were mixed in 200 mL of CH2Cl2. The reaction mixture was stirred overnight at room temperature. The precipitated urea DCC byproduct was filtered off and washed with a small volume of CH2Cl2. The crude product was concentrated using a rotavapor and precipitated into 1 l of cold hexane. After one night in the fridge, the product is filtered and washed with cold hexane to give the product as a white powder. Yield: 92 %. 1H-RMN (300 MHz, CDCl3): δ (ppm): 1.12 (s, 6H, G), 3.69 (d, J=11.6 Hz, 4H, F), 4.66 (d, J=11.7 Hz, 4H, F´), 5.47 (s, 2H, E), 7.34 (m, 6H, A, B), 7.45 (m, 4H, C). 13C-RMN (75 MHz, CDCl3): δ (ppm): 16.9 (G), 44.2 (H), 73.2 (F, F´), 102.1 (E), 126.3 (C), 128.2 (B), 129.1 (A), 137.6 (D), 169.1 (I). IR (cm-1, KBr): 2865 (C-H st), 1816 (C=O st sim), 1746 (C=O st as). 40 -Synthesis of P/B-2 OOO 67 O 98 O 98 O O J O H O O H O O A B C D D E EF G F G H I´ I L M N N P OP K Dry Pluronic F-127 (10.0 g, 0.79 mmol), DMAP (0.12 g, 0.98 mmol) and benzylidene2,2-bis(oxymethyl)propionic anhydride (2.0 g, 4.68 mmol) were dissolved in 20 mL of CH2Cl2. The mixture was stirred at room temperature overnight under argon blanket. The excess of anhydride was quenched by adding 7 mL of methanol. The mixture was stirred while 6 hours and the crude was precipitated in 1 L of cold diethyl ether. After a night in fridge the product was isolated by filtrating and washing with diethyl ether, as a white powder. Yield: 97 %. 1H-RMN (400 MHz, CDCl3): δ (ppm): 1.04 (s, 6H, J), 1.13 (m, 201H, A), 3.37-3.82 (m, ~1100H, B, C, D, E, F, I´), 4.35 (m, 4H, G), 4.66 (d, J=11.6 Hz, 4H, I), 5.44 (s, 2H, L), 7.32 (m, 6H, P, O), 7.42 (m, 4H, N). 13C-RMN (300 MHz, CDCl3): δ (ppm): 17.3, 17.4 (A), 17.9 (J), 42.4 (K), 64.2 (G), 68.568.6-69.1 (F), 70.5 (D, E), 72.973.3 (C, I, I´), 75.175.375.5 (B), 101.7 (L), 126.2 (N), 128.2 (Q), 128.9 (P), 137.9 (M), 173.9 (H). -Synthesis of P/OH-4 OOO 67 O 98 O 98 O HO J HO OH OH O O A B C D D E EF G F G H I I K In a flask, compound P/B-2 (10 g, 0.77 mmol) was dissolved in 200 mL of EtOAc. Once P/B-2 is dissolved, 10 % (w/w) Pd/C was added. After three vacuum-argon cycles, the reaction mixture was stirred at room temperature in hydrogen atmosphere 41 overnight. The Pd/C was filtered off with Celite® and the filtrate was evaporated to give the product as white solid. Yield: 93 %. 1H-RMN (400 MHz, CDCl3): δ (ppm): 1.11 (s, 6H, J), 1.13 (m, 201H, A), 3.37-3.82 (m, ~1100H, B, C, D, E, F, I), 4.34 (m, 4H, G). 13C-RMN (300 MHz, CDCl3): δ (ppm): 17.1 (J), 17.317.4 (A), 49.5 (K), 63.2 (G), 67.3 (I), 68.468.568.7 (F), 70.5 (D, E), 72.973.3 (C), 75.175.375.5 (B), 175.6 (H). -Synthesis of P/NP-4 In a flask, P/OH-4 (3 g, 0.233 mmol, 1 eq) was dissolved in 20 mL of dry CH2Cl2, then, 0.8 mL of dry pyridine and p-nitrophenyl chloroformate (0.75 g, 3.76 mmol, 16 eq), were added to the reaction flask under argon blanket and stirring. The reaction mixture was stirred at room temperature for 24 h. The crude was redisolved with 50 mL of dichloromethane and extracted twice with a 1M NaHSO4 solution (2x30 mL) and with a NaCl saturated solution (1x30 mL), dried with MgSO4, filtered, and evaporated in rotavapor to reduce the volume. Then, the product was precipitate in 300 mL of cold diethyl ether, placed in the fridge overnight, filtered and washed with cold diethyl ether. Yield: 79 %. 1H-RMN (300 MHz, CDCl3): δ (ppm): 1.13 (m, 201H, A), 1.38 (s, 6H, J), 3.37-3.85 (m, ~1100H, B, C, D, E, F), 4.34 (m, 4H, G), 4.47 (d, J= 9 Hz, 4H, I), 4.58 (d, J= 9 Hz, 4H, I´), 7.37 (m, 8H, M), 8.26 (m, 8H, O). 13C-RMN (75 MHz, CDCl3): δ (ppm): 17.417.5 (A), 17.7 (J), 46.5 (K), 64.5 (G), 68.668.7-69.2 (F), 70.7 (D, E, I, I´), 72.973.5 (C), 75.275.475.6 (B), 121.7 (M), 125.3 (O), 145.5 (P), 152.1 (L), 155.2 (N), 171.5 (H). 42 -Synthesis of P/C-4 OOO 67 98 O 98 O A B C D E F G N M O O O O O O O O N H N H O O H N H N O O H I K J L OH OH HO HO In a flask, P/NP-4 (1.6 g, 0.118 mmol, 1 eq) was dissolved in 10 mL dry dichloromethane under argon blanket and stirring. Ethanolamine (0.043g, 0.704 mmol, 6 eq) along with 2 mL of dry dichloromethane, were added to the reaction flask. The reaction mixture was stirred for 24 h at room temperature, next, the crude was precipitated into 200 mL of cold diethyl ether and placed in the fridge overnight. The product was filtered and washed with cold diethyl ether to obtain the product as a white powder. Yield: 90 %. 1H-RMN (300 MHz, CDCl3): δ (ppm): 1.13 (m, 201H, A), 1.23 (s, 6H, J), 3.29 (m, 8H, M), 3.39-3.87 (m, ~1100H, B, C, D, E, F, N), 4.26 (m, 12H, I, G). 13C-RMN (75 MHz, CDCl3): δ (ppm): 17.1 (A), 43.3 (K), 46.4 (M), 61.2 (N), 65.6 (I) 66.8 (G), 68.6 (F), 70.2 (D, E), 72.673.08 (C), 74.875.2 (B), 156.35 (L), 172.85 (H). -Synthesis of P/A-4 O P OOO 67 98 O 98 O A B C D E F G N M O O O O O O O O N H N H O O H N H N O O H I K J L O O O O O O O O Q, Q' In a flask, P/C-4 (1.2 g, 0.091 mmol, 1 eq) was solved in 10 mL of dry dichloromethane (or dry THF) under argon blanket and stirring, then the inhibitor, 4-methoxyphenol was added in excess (300-850 mg). Once the mixture was solved, the flask was placed in an ice bath and TEA (0.162 g, 1.6 mmol, 17.6 eq) was added dropwise under inert atmosphere. Next, acryloil chloride (0.132 g, 1.46 mmol, 16 eq) was added dropwise and also under inert atmosphere. The reaction mixture was stirred at room temperature 43 for 48 hours in the dark. The solution was passed through a neutral alumina column and the filtrate was dried with Na2CO3 during 2 hours, filtered and evaporated to reduce the volume. Finally the product was precipitate in cold diethyl ether (200-300 mL) and stored in the fridge overnight. The product was filtered and washed with cold diethyl ether. 5.3.-TIM and DSC procedures - TIM procedure First, the chosen concentration samples were prepared in solution with PBS (10 mM, pH =7.4), 100 μl, inside a 2 mL vial. To obtain a good solution the samples were placed at 4 º C overnight and in the dark. Then, the samples were heated with a heater block (speed 1 ºC/min.). During the heating process, the state of the samples was visually followed to determine at what temperature, indicated by the heater block, the transition from sol state to gel state took place for each sample. The LCST was defined as the temperature when the material does not flow for at least 10 seconds, when the vial is upside down. - DSC procedure The samples were introduced inside of airtight capsules, and the applied method for all samples was the same: to heat the sample from -10 °C to 50 °C, following by a cooling process from 50 °C to -10 °C with a ramp of 5 °C/min. 5.4.-Photopolymerized macroscopic hydrogel obtention To prepare the photopolymerized hydrogels, the obtained products were prepared at selected concentrations, by dissolving them in 100 μL of a previously prepared PBS 10 mM solution containing 0.1% w/v of photoinitiator Irgacure 2959. IRGACURE 2959 is a highly efficient non yellowing radical photoinitiator for the UV curing of systems comprising of unsaturated monomers and prepolymers. It is especially suited where low odor is required and for use in water borne systems based on acrylate or unsaturated polyester resins29. 44 These solutions were stored overnight at 4°C and in darkness to ensure a good and homogeneous dissolution of the products. Subsequently, the gels are formed by the effect of temperature. To do this, a hotplate was used (see Figure 28). Upon the hotplate, a cylindrical holder of 6 mm diameter by 3 mm high, is positioned on a glass slide. When the achieved temperature by the hotplate was 37°C, the cold solution prepared is poured into the holder. This process must be carried out keeping and manipulating the samples in cold conditions, in order to prevent the gel formation prematurely. Once the gel was formed within the holder, it was exposed to ultraviolet radiation (365nm) at 8 cm distance for 10 minutes to fix the structure. After the 10 minutes of exposure to the UV light, the photopolymerized hydrogels are demoulded and used for its morphological characterization by SEM and for the degradation studies. 5.5.-Swelling and degradation procedure The procedure method was as follows:  To weigh the freshly prepared photopolymerized hydrogel at certain concentration. Defining Wo as the initial weight of the hydrogel.  Each sample was incubated in 2mL of PBS (10 mM, pH=7.4) at 37 °C.  At constant intervals of time, equal for all samples, each one was weighed. For this purpose, the sample was dried prior to weighing by removing excess of c a c Figure 28. Hydrogel’s photopolymerization system. a) hotplate at 37 ºC. b) cylindrical mould. c) UV lamp. a b 45 PBS, subsequently, another 2 mL of new PBS in the same conditions were added to recover the experimental swelling conditions. 5.6.-Photopolymerized nanogel obtention. For obtaining the nanogels, two solutions were prepared separately  First, it is necessary to prepare a solution containing 0.1% (w/v) of the photoinitiator Irgacure 2959 in distilled water. (Solution A)  Secondly, a solution containing the product P/A-2, 10 % in (w/v), dissolved in 600 μL of solution A, this one was placed overnight at 4ºC in darkness to ensure an homogeneous dissolution of the material. (Solution B) Then, both solutions were filtered with a 0.20 μm filter, and taken to a final volume of 5 ml (Solution C), where the P/A-2 concentration was 0.77 % wt. Then, the solution C was poured in a clean glass container where the photopolymerization process will be carried out. The solution was exposed to ultraviolet radiation (365nm) with 8 cm distance between the lamp and the sample, for 10 minutes, at room temperature. Once the structure of the nanogel has been fixed, the photopoymerized solution is placed inside a membrane [cellulose ester (CE), MWCO300 000 with a nominal pore size of 35 nm], and dialyzed for 24h at 4ºC in a 2 L vessel. The aim of the dialysis process is to purify the nanogel, mainly by eliminating all the possible particles which could be within the nanogel solution, chiefly the presence of free monomers. Once the dialysis process was finished, the samples are again filtered with a single use 0.20 μm filter and prepared to be characterized by TEM, SEM and DLS. 46 5.7.-MSCs and HeLa cell culture and sample preparation.  MSCs First MSCs were grown in alfa-MEM (minimum essential medium) with FGF (fibroblast growth factor) and 10 % of FBS (fetal bovine serum). Media was removed each 3 or 4 days, and cells were cells trypsinized after reaching 80% confluence. To prepare the samples, Pluronic F127, P/C-2 or P/A-2, in its solid state, were sterilized by UV light for an hour. Next, these sterilized compounds were dissolved in an also sterilized DMEM, with or without photoinitiator Irgacure 2959 (0.1 %). DMEM is a modification of Basal Medium Eagle (BME) that contains a four-fold higher concentration of amino acids and vitamins, as well as additional supplementary components.To ensure a good solubilization, samples were left overnight in the refrigerator at 4 ° C, tightly closed. For 2D assays, 10000 cells/well were seeded in a 96 well-plate. Cells were covered by 100μL of polymeric solution (Pluronic 127, P/C-2 or P/A-2), and incubated for 5 minutes at 37 °C to reach the physical hydrogel. Then, the photopolymerization process was carried out when needed, by exposing the 96well-plate to UV light for 10 minutes at 8 cm distance. After it, 100 μL of freshly medium was added. In the case of 3D assays, the process was similar but previously, cells were dispersed into the polymeric solution, and then the assembly was transferred to the 96 well-plate.  HeLa cells HeLa cells were seeded at a density of 1-3 x103 cells/well in a 96 well-plate with DMEM. The P/A-2 PDF nanogel, in this case, was prepared as explained in Experimental section 5.6, then lyophilized and dissolved again in sterile DMEM, 1mg/mL. Upon 24h incubation, media was removed and fresh media was added to the control wells and the P/A-2 nanogel diluted in media at different concentrations (0.25, 0.5 and 1 mg/mL) was added to the rest of the wells. 47 Media was removed after 24, 48 and 72h of incubation with the P/A-2 PDF nanogel, and fresh media was added to all the wells (control and sample wells); then a 10% of the media volume of Alamar Blue solution (Invitrogen by Life Technologies, Thermo Fisher Scientific, Spain) was added. After 2h incubation at 37ºC, fluorescence was read at 530/590 (excitation/emission) on a Synergy HT (BioTek, USA) plate reader. 6.-Conclusions  Pluronic F127 allows to prepare thermosensitive and photopolymerizable hydrogels by the incorporation of hydroxyl and acrilate terminal groups.  The photopolymerization technique can be successfully applied for the preparation of both the macroscopic molded hydrogels and nanostructured hydrogels (nanogels).  Concerning the macroscopic hydrogel: o The internal morphology, characterized by SEM, reveals a pore size distribution from 4 to 12 μm. o The materials exhibit a total degradation in 92 days. o Functionalized derivatives offer better cell viability than Pluronic F127 in the 2D assays.  Concerning the nanostructured hydrogel: o Functionalized derivatives offer improved CMC than Pluronic F127. o The morphological characterization by TEM and SEM, have confirmed the micellar structure of the nanogels in water, and the possibility of its drying and further re-dissolution. 7.-References 1. 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Zentner and B. Jeong, US Pat., 6117949, 2000. c)A. Chenite, C. Chaput, D. Wang, C. Combes, M. D. Buschmann, C. D. Hoemann, J. C. Leroux, B. L. Atkinson, F. Binette and A. Selmani, Biomaterials, 2000, 21, 2155–2161. d) B. H. Lee, Y.M. Lee, Y. S. Sohn and S. C. Song, Macromolecules, 2002, 35, 3876–3879. 9. Li, Y.; Rodrigues, J.; Tomas, H., Injectable and biodegradable hydrogels: gelation, biodegradation and biomedical applications. Chemical Society Reviews 2012, 41 (6). 10. (a) Balakrishnan, B.; Banerjee, R., Biopolymer-Based Hydrogels for Cartilage Tissue Engineering. Chemical Reviews 2011, 111 (8), 4453-4474; (b) Naveena, N.; Venugopal, J.; Rajeswari, R.; Sundarrajan, S.; Sridhar, R.; Shayanti, M.; Narayanan, S.; Ramakrishna, S., Biomimetic composites and stem cells interaction for bone and cartilage tissue regeneration. Journal of Materials Chemistry 2012, 22 (12). 11. Center for Disease Control and Prevention, statistics data, 2003. National Health Interview Survey (NHIS) Statistics. 12. T.Furukawa, D.R. Eyre and M.J. Glimcher, J. Bone Joint. Surg. Am., 1980, 62, 79-89. 13. Khandare, J.; Calderon, M.; Dagia, N. M.; Haag, R., Multifunctional dendritic polymers in nanomedicine: opportunities and challenges. Chemical Society Reviews 2012, 41 (7), 28242848. 14. Kabanov, A. V.; Vinogradov, S. V., Nanogels as Pharmaceutical Carriers: Finite Networks of Infinite Capabilities. Angewandte Chemie-International Edition 2009, 48 (30), 5418-5429. 15. Lee, W.-C.; Li, Y.-C.; Chu, I. M., Amphiphilic Poly(D,L-lactic acid)/Poly(ethylene glycol)/Poly(D,L-lactic acid) Nanogels for Controlled Release of Hydrophobic Drugs. Macromolecular Bioscience 2006, 6 (10), 846-854. 16. G.Yu, X. Yan, C.Han, F.Huang. Characterization of supramolecular gels. Chem.Soc.rev.,2013, 42, 6697. 17. Nie, S. F.; Hsiao, W. L. W.; Pan, W. S.; Yang, Z. 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V 13C-NMR IR 1728,1 05001000150020002500300035004000 cm-1 P/A-2 K H M L A I F G J D,E B C VI ⱷ P/C-4 1H-NMR 13C-NMR OOO 67 98 O 98 O A B C D E F G N M O O O O O O O O N H N H O O H N H N O O H I K J L OH OH HO HO A J M B+C+D+E +F+N I+G A K M N H L B C VII APPENDIX 2 ⱷ DSC Pluronic F127 16.5 % wt 14.16 mg sample 18 % wt 14.32 mg sample 20 % wt 3.54 mg sample 23 % wt 9.82 mg sample 25 % wt 15.95 mg sample VIII P/C-2 16 % wt 5.73 mg sample 18 % wt 11.60 mg sample 20 % wt 3.61 mg sample 23 % wt 4.41 mg sample 25 % wt 4.97 mg sample IX P/A-2 16 % wt 15.59 mg sample 18 % wt 11.45 mg sample 20 % wt 6.30 mg sample 23 % wt 17.08 mg sample 25 % wt 16.50 mg sample X ⱷ Materials and methods Materials: - All the products where purchased from Sigma-Aldrich and Acros Organics. -The dialysis membrane was purchased from Spectrum. General methods: - 1H-NMR and 13C NMR spectra were recorded on a Bruker AV-400 (operating at 400 MHz for 1H and 100 MHz for 13C) and on a Bruker AMX300 (operating at 300 MHz for 1H and 75 MHz for 13C). CDCl3 was used as solvent, chemical shifts are given ppm relative to TMS, and the solvent residual peak was used as internal standard. - The infrared spectra of all the complexes were obtained with a Bruker Vertex 70 in ATR mode model MKII Golden Gate Single Reflection ATR System from Specac. - Differential scanning calorimetry (DSC) was performed using a DSC Q20 V24.10 Build 122 from TA Instruments. - Mass Spectrometry was performed using an ESI Brüker Esquire 300+, a MALDI+/TOF Brüker Microflex system. - SEM analyses were performed with a SEM Inspect F50 with gold or platinum coated samples, at the Laboratory of Advanced Microscopy (LMA) of the INA (Nanosciences Institute of Aragon). - TEM measurements were performed using a TECNAI G20 (FEI COMPANY), 200 kV, at the Laboratory of Advanced Microscopy (LMA) of the the INA (Nanosciences Institute of Aragon). Samples were prepared on holey carbon film 300 Mesh Cu (50) from Agar Scientific. - DLS measurements were performed using a Brookhaven 90 Plus Particle Analyzer. XI - GPC measurements were performed using a Waters e2695 Alliance liquid chromatography system equipped with a Waters 2424 evaporation light scattering detector, Styragel® columns HR1 from Waters. Measurements were performed in THF with a flow of 1mL min-1 using polystyrene (PS) narrow molecular weight standard. - Fluorescence measurements were performed in a Perkin Elmer LS 55 fluorescence spectrometer. - Cells images were taken at a fluorescence inverted microscope model Olympus IX81 at CIBA (Aragon Biochemical Research Center). ⱷ Staining for cell viability tests  LIVE/DEAD® Viability/Cytotoxicity Kit Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells . EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells. EthD-1 is excluded by the intact plasma membrane of live cells. The determination of cell viability depends on these physical and biochemical properties of cells. Cytotoxic events that do not affect these cell properties may not be accurately assessed using this method. Background fluorescence levels are inherently low with this assay technique because the dyes are virtually non-fluorescent before interacting with cells.  AlamarBlue® The assay is based on the ability of viable, metabolically active cells to reduce resazurin to resorufin and dihydro-resorufin. This conversion occurs intracellularly, where the oxidized form of the resazurin enters the cytosol and is converted to the reduced form XII by mitochondrial enzyme activity by accepting electrons from NADPH, FADH, FMNH, NADH as well as from numerous cytochromes. The reduction related to growth causes the resazurin to be converted from the oxidized (or non-fluorescent) blue form to the reduced (fluorescent) red form. Since Resazurin is not-toxic to cells and is stable in culture media, continuous measurement of cell proliferation in vitro can be achieved. Toxic compounds that impair cell viability and proliferation also affect the capacity to reduce resazurin, and the rate of dye reduction is directly proportional to the number of viable cells present.