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PAMAM-based Dendrimers for Diverse Biomedical Applications Author Rebeca González Pastor Directors María del Pilar Martín Duque. Araid researcher at I+CS Jose Luis Serrano Ostáriz. Zaragoza University/INA Faculty of Sciences Zaragoza, September 2012 Master in Nanostructured Materials for Nanotechnology Applications 2 TABLE OF CONTENTS Summary......................................................................................................4 PART I: INTRODUCTION.......................................................................5 1. GENE AND CELL THERAPIES........................................................................... 5 1.1 Development and evolution..................................................................................... 5 1.2 Vectors for Delivery................................................................................................ 7 1.2.1. Viral and cellular Vectors:.............................................................................. 8 1.2.2. Non-Viral Vectors.......................................................................................... 9 2. MICROBIAL RESISTANCE................................................................................ 10 3. DENDRIMER-BASED VECTORS...................................................................... 10 3.1 Cationic Polymers ................................................................................................. 10 3.2 Dendritic architectures........................................................................................... 11 3.3 Synthesis and Features .......................................................................................... 13 3.4 Physicochemical Properties................................................................................... 14 3.5 PAMAM and PAMAM-modified dendrimers ...................................................... 14 4. OBJECTIVES IN THIS PROJECT..................................................................... 16 PART II: MATERIALS AND METHODS............................................17 1. SYNTHESIS OF mPEG 2000 -G3-PAMAM............................................................ 17 2. STRUCTURAL ANALYSIS OF mPEG 2000 -G3-PAMAM ................................. 18 2.1 Material Characterization...................................................................................... 18 2.2 Assessment of dendrimer degradation................................................................... 18 3. DNA-DENDRIMER COMPLEX FORMATION ............................................... 18 3.1 Plasmid DNA Preparation..................................................................................... 18 3.2 Gel Retardation Assay........................................................................................... 20 4. EVALUATION OF BACTERIAL GROWTH.................................................... 20 4.1 Bacterial Preparations............................................................................................ 20 4.2 Bacterial Assays .................................................................................................... 21 4.2.1. LB plate assay............................................................................................... 21 4.2.2. LB broth assay.............................................................................................. 21 4.2.3. LB microplate assay..................................................................................... 22 PAMAM-based Dendrimers for Diverse Biomedical Applications 3 5. EVALUATION OF CELL GROWTH................................................................. 22 5.1 Culture of Cell Lines............................................................................................. 22 5.2 Viability Assays..................................................................................................... 24 5.2.1. Crystal Violet method................................................................................... 24 5.2.2. Alamar Blue method..................................................................................... 25 5.2.3. MTT method................................................................................................. 26 5.2.4. Interference Tests ......................................................................................... 27 5.2.5. Trypan Blue method..................................................................................... 27 5.3 Gelatin Assay......................................................................................................... 28 5.4 Evaluation of Cellular Alterations......................................................................... 28 5.4.6. Analysis of Cell Cycle and Immunophenotyping ........................................ 28 5.4.7. mMSCs Adipogenic Differentiation............................................................. 29 PART III: RESULTS AND DISCUSSION ............................................32 1. STRUCTURAL ANALYSIS OF mPEG2000-G3-PAMAM............................... 32 2. DNA-DENDRIMER COMPLEX FORMATION ............................................... 34 3. EVALUATION OF BACTERIAL GROWTH.................................................... 35 4. EVALUATION OF CELLULAR GROWTH AND ALTERATIONS.............. 38 CONCLUSIONS........................................................................................46 Acknowledgments......................................................................................47 References..................................................................................................47 Master in Nanostructured Materials for Nanotechnology Applications 4 Summary There is a great need for new treatments in certain pathologies such as cancer or resistant infections. Among some of the most promising novel therapies is gene therapy, but it faces serious problems to overcome before using viral vectors into the clinic. To solve some of those problems viral gene therapy vectors are recently combined with stem cells in cell therapy approaches. However, as they are complicated cells to grow with standard tissue culture methods and culture media, this approach has to be improved previously to move into manufacturing for clinical trials. Synthetic gene-delivery agents such as polymers, although safer than viruses, generally do not possess the required efficacy. Dendrimers are three-dimensional, highly branched, synthetic polymers with a well-defined chemical structure. In particular, PAMAM (poly(amidoamine)) dendrimers have been extensively investigated for their biological applications and they have shown clinically-relevant carrier properties that could be used for gene or cell therapy purposes. This is facilitated by controlling charge and functionality through the choice of peripheral groups, and size through generation number and PEGylation. PEG (poly(ethylene glycol)) reduces toxicity, improves water solubility, decreases enzymatic degradation, and leads to improved biodistributions characteristic. Moreover, it has been shown that PAMAM dendrimers can facilitate antibiotic localization also inside bacteria, and so, they can be used to enhance the antimicrobial activity. Also, they could be highly toxic to certain bacterias themselves by interacting and promoting disruption of the cytoplasmatic membrane. By controlling the size and PEGylation degree of the PAMAM dendrimers we can modulate the interactions with the bacterial surface and their killing activity, lowering the cytotoxicity to the host cells. Therefore, PAMAM dendrimers appear as promising antimicrobial agents that are less likely to induce bacterial resistance compared to standard antibiotics. Here we study the biomedical properties of PAMAM-derived dendrimers on their influence on the bacterial and cellular growth. Our results show that, although the PAMAM dendrimer completely covered with PEG diminished the pseudomonas growth and it has potential to be employed as an antibacterial agent, in the case of mesechymal stem cells, the cell growth is increased without altering their pluripotenciality patterns, and it could be of great help for the future cell therapy approaches. PAMAM-based Dendrimers for Diverse Biomedical Applications 5 PART I: INTRODUCTION 1. GENE AND CELL THERAPIES 1.1 Development and evolution Gene therapy aims to treat both genetic and infectious diseases by restoring, modifying or enhancing cellular functions through the introduction of new genetic material into the appropriate cells in the body. While traditional drug therapies involve the administration of chemicals that have been manufactured outside the body, gene therapy takes a very different approach: directing a patient's own cells to produce and deliver a therapeutic agent. Gene therapy protocols were originally designed to correct inheritable disorders, such as cystic fibrosis, Gaucher's disease, and Duchenne muscular dystrophy (Dekker,3). Of the two potential strategies for gene therapy, the ex-vivo and the invivo, the exvivo (cell-based delivery) strategy allows a wider range of therapies. In this type, cells (especially stem cells) would be extracted from patients, transfected with the therapeutic gene, grown in culture, and reimplanted in the patient. In the in-vivo (direct delivery) approach, genes are administered directly into the patient to transduce the cells in-vivo; this delivery method is fairly imprecise and limited to the specific types of human cells that the vector can reach (Vetrini, et al,9). Figure 1.1. Strategies for Delivering Therapeutic Transgenes into Patients (Dekker,3). Master in Nanostructured Materials for Nanotechnology Applications 6 The first approved gene therapy clinical protocol began in September 1990, using retroviral vectors to introduce copies of the adenosine deaminase gene into T cells from a patient with adenosine deaminase deficiency. However, clinical progress has been slow. In 2002, over 600 clinical protocols have been approved worldwide, incorporating over 3500 patients (The Journal of Gene Medicine). A major setback for the field occurred in September 1999, when a man died from a massive immune reaction to the Ad5 vector that was used to deliver a deficient gene. Moreover, in that same year three patients suffering from a severe combined immunodeficiency disorder (SCID-XI) developed leukemia as a result of the gene-transfer procedure. This trial reflects the path that gene therapy followed: success, but with some complications (Dekker,3). Although it was conceived as a therapeutic modality for genetic disorders, over 60% of these trials have been directed to the treatment of cancer, reflecting the need for novel therapies in this field (The Journal of Gene Medicine). Cancer represents an enormous biomedical challenge for gene therapy and drug delivery. Tumors are caused as a consequence of mutations in the genome by addition, deletion or change in chromosomal pattern. Ideally, the goal of any cancer therapy is to selectively target and destroy diseased tissue while sparing the surrounding healthy tissue. Over the past three decades, cancer diagnosis, surgery and treatment have improved greatly, but traditional therapies (chemotherapy and radiotherapy) are still insufficient (Agarwal, et al,10). In an attempt to solve this problem, new and more specific therapies exclusively targeted against cancer cells are currently being developed (Vetrini, et al,9; Pandita, et al,11; Li, et al,12; Niidome, et al,13). One of the key problems in gene therapy is the efficient delivery of therapeutic transgenes to pathological sites, upon systemic injection of the vector (Flint,1; Dekker,3). In this context, the ideal vector should show high expression levels, tissue selectivity, and ability to avoid immune response after vector re-administration or over long time periods. Amongst all the commonly used vectors, Figure 1.2. Indications addressed by Gene Therapy Clinical Trials (The Journal of Gene Medicine, 2012) PAMAM-based Dendrimers for Diverse Biomedical Applications 7 none meet the mentioned requirements. For that reason, development of new vectors is pivotal for the development of gene therapy (Wolinsky, et al,6; Vetrini, et al,9; Agarwal, et al,10; Li, et al,12; Niidome, et al,13; Chen, et al,14). Tumours are composed of both malignant and normal cells. The “benign” cell-types include endothelial cells forming blood vessels, infiltrating inflammatory cells, and stroma. Stromal components provide factors and structural support for malignant cells. The formation of tumour stroma closely resembles wound healing and scar formation that increase proliferation of mesenchymal stem cells (MSCs) (Dekker,3; Barry,7; Pandita, et al,11). Some groups have hypothesized that exogenously administrated MSCs would preferentially go to damaged areas independently of the pathology, and in the case of tumours they would engraft at the tumor site and contribute to the population of the tumour stroma (Santos, et al,15). Based on that hypothesis, MSCs have been recently used as vectors to carry therapeutic genes specifically into tumoral areas. 1.2 Vectors for Delivery As the success of gene therapy is highly dependent on the delivery vector, research in this field has been focused on the development of a suitable delivery system. Two most important criteria for a viable gene delivery vector are safety and efficacy: it must not elicit an immune response, must not be infectious and must not cause mutagenesis due to improper gene insertion, while incorporating sufficient functionality to overcome the multitude cellular and extracellular barriers to gene transfer. The first barrier is cellular uptake, where the vector must associate with the cell surface; once inside the cell, the gene must be released from the endosomes and finally it must be able to overcome the nuclear membrane and enter the nucleus. Currently the most common and efficient delivery systems are viruses. Major safety concerns, however, such as high toxicity and immunogenicity, have limited their general use. Investigators of non-viral delivery systems have shown marked improvements in efficiency (Vetrini, et al,9; Niidome, et al,13). Figure 1.3. Vectors used in Gene Therapy Clinical Trials (The Journal of Gene Medicine, 2012) Master in Nanostructured Materials for Nanotechnology Applications 8 1.2.1. Viral and cellular Vectors: Viral gene delivery systems exploit the natural mechanisms evolved by different viruses for cellular entry and gene transfer. However, of the virus-based vector systems developed thus far, none are optimal and each system displays advantages and disadvantages characteristic of each virus (Dekker,3; Marvaniya, et al,4; Wolinsky, et al,6; Agarwal, et al,10; Tomalia, et al,16; Flint,17; Biasco, et al,18). In general, the genetic material of the virus is manipulated to carry a copy of the desired gene as well as a promoter. The promoter determines whether the gene functions within the cell or not. A drug usually activates the promoter, which in turn, activates the gene. The activated gene is then translated into proteins. The safety concerns (retention in the liver, immunogenicity, oncogenicity, etc.) and difficult large-scale production limits the usefulness of recombinant viral vectors (Flint,1; Dekker,3; Biasco, et al,18). Mammalian cells (fibroblasts, hepatocytes, mesothelial cells, myoblast, neuronal cells, etc) have been proposed as new vector systems, because of their properties with regard to immune recognition and toxicity. Some studies suggest that bone marrow cells could differentiate widely in different tissues and form hepatic cells, cardiomyocytes, neurones, etc (Orkin, et al,19; Morrison,20). Mesenchymal stem cells (MSCs) have been studied as vehicles for gene and cellular therapy because they are easy to extract from bone marrow (Flores-Figueroa, et al,21; 22) and to recover organs with just a small population expressing the corrected gene. That is why this new field has been named “regenerative therapy” (Pomerantz, et al,23). There are hundreds of clinical trials ongoing using MSCs under the common hypothesis that MSCs are able to cure and regenerate the damaged areas independently on the pathology of study (Bobis, et al,24). Figure 1.4. Retroviral life cycle (Flint,1). PAMAM-based Dendrimers for Diverse Biomedical Applications 9 Some groups hypothesized that exogeneous administered mesenchymal stem cells (MSCs) would contribute to tumour stroma formation and thus could transport substances in a therapeutic context. Tumour stroma formation can be considered functionally analogous to the scar formation process undergone following tissue damage, both processes involving high MSC proliferation (Mackenzie, et al,25). Therefore, such cells could be used as a virus transporter directed specifically to different pathologies. 1.2.2. Non-Viral Vectors Besides virus-mediated gene-delivery systems, there are several nonviral options for direct gene delivery that are attractive alternatives for improved safety, greater flexibility and ease of manufacturing. The simplest method is the direct introduction of therapeutic DNA into target cells. A number of barriers must be overcome: it must be protected from degradation, it must cross the cell membranes and when chemical methods are used, the complexes formed by the DNA and by the non-viral vectors must be released from the formed endosomes and decomplexation must occur. Also, it can only be done in certain tissues and requires large amounts of DNA (Marvaniya, et al,4; Wang, et al,26; Maruyama-Tabata, et al,27; Fant, et al,28; Qi, et al,29). Other approaches use other vectors to transport DNA to the cell: synthetic nonviral vectors. Synthetic vectors are more versatile: their chemical and physical properties, for instance, allow the packing of a large variety of gene lengths, in contrast to the capsid restricted capacity of viruses. Although synthetic vectors are now routinely used in laboratories, their very low delivery efficiency remains prohibitive for their use in gene delivery (Marvaniya, et al,4; Li, et al,12; Niidome, et al,13; Voulgarakis, et al,30). One major approach in nonviral gene therapy is based on cationic polymers. In the following pages we will focus on an example of this type of polymer, a modified polyamidoamine (PAMAM) dendrimer. Master in Nanostructured Materials for Nanotechnology Applications 16 4. OBJECTIVES IN THIS PROJECT The specific objectives to reach will be: 1. Characterize the formation of complexes and stability in cell culture conditions of the dendrimeric form. 2. To test the toxicity of the dendrimer through in vitro experiments to be complexed on a near future to therapeutic DNA or adenoviruses to suffer of EPR effect and will target specifically tumors. 3. To study the growth changes on the MSCs culture conditions when incubated with PAMAM-based dendrimeric forms and how they could make an influence on the surface markers or pluripotency status of the cell 4. To test the potential antimicrobial efficiency of the dendrimer. PAMAM-based Dendrimers for Diverse Biomedical Applications 17 PART II: MATERIALS AND METHODS 1. SYNTHESIS OF mPEG 2000 -G3-PAMAM The commercial amine-terminated G3 PAMAM dendrimer with an ethylenediamine core (20 wt% in methanol, Sigma-Adlrich, Spain) was vacuum-dried to remove traces of methanol. Poly (ethylene glycol) methyl ether (PEG, Mn=2000) was first thoroughly dried and then dissolved in tetrahydrofuran (THF) and triethylamine (TEA) (both distilled and dried). PEG was firstly activated via a reaction with p-nitrophenyl chloroformate chloride, which was slowly added to the mixture and stirred for 24h at room temperature (a calcium chloride tube is used to prevent any moisture from getting into the reaction vessel). The hydroxyl groups of methylated PEG reacted with the reagent and eliminated a molecule of HCl, as shown in Figure 2.1. The salt was filtered off and the filtrate was concentrated and then added dropwise to diethyl ether to precipitate as activated mPEG p-nitrophenyl chloroformate. The precipitate was dissolved in THF again and it was purified by reprecipitation from THF into diethyl ether. Alternatively, the purification could be carried out in a Sephadex-LH20 column using MeOH as eluent. Next, pegylation was performed by linking activated p-nitrophenyl chloroformate PEG (4-NPC-PEG) with G3 PAMAM dendrimer. A solution of the dendrimer was dissolved in dimethyl sulfoxide and mPEG p-nitrophenyl carbonate was added to the solution at 4ºC and then stirred for 72h at room temperature. The reaction mixture was evaporated using a rotatory evaporator. The obtained product was dissolved in distilled water and filtrated in order to eliminate the low molecular weight reagents. The solid product was characterize by 1 H MNR and kept protected from moisture and light. The synthesis was carried out with the collaboration of Ramón Cervera, from the Liquid Crystals and Polymers Group (University of Zaragoza). Fig 2.1. Diagram showing the synthesis of mPEG-G3-PAMAM dendrimer (Bai, et al,8). Master in Nanostructured Materials for Nanotechnology Applications 18 2. STRUCTURAL ANALYSIS OF mPEG 2000 -G3-PAMAM 2.1 Material Characterization The stoichiometry of mPEG 2000 -G3-PAMAM was established by 1H NMR. Since each PAMAM dendrimer conjugate was not unimolecular, characterization represented the average value from its polymeric distribution. Spectra were recorded on a Bruker AVANCE 400 spectrometer operating at 400 or 300 MHz and using DMSO and d6/D2O as solvents. Chemical shifts are given in ppm relative to TMS, and the solvent residual peak was used as internal standard. Ten milligrams of the dendrimer were dissolved in DMSO or deuterated solvent to perform the analysis. MALDI mass spectra were obtained using 2,5-dihydroxybenzoic acid (DHB) and 2,4,6-trihydroxyacetophene (THAP) as matrices on a AUTOSPEC Vacuum General. Average molecular weights were calculated from the mass range encompassing the desired major peak. Fourier transform infrared (FTIR) spectra were recorded on a Mattson Genesis II FTIR. Samples were scanned between 4,000 and 700 cm -1 at a resolution of 1cm -1 . 2.2 Assessment of dendrimer degradation mPEG 200 0-G3-PAMAM was dissolved in DMSO-d6/D 2 O (10mg/ml) or left solid. Some of the aliquots were kept protected from light but others were exposed to light at 4ºC, room temperature and 37ºC for periods of time ranging from 24-72h. The solid samples were dissolved in DMSO-d6/D 2 O (10mg/ml) right before the analysis. The integrity of mPEG 2000 -G3-PAMAM was established by 1 H NMR using the same equipment and conditions as previously described (Bruker AVANCE 400). 3. DNA-DENDRIMER COMPLEX FORMATION 3.1 Plasmid DNA Preparation The pEGFP plasmid (Addgene, USA) was amplified in chemically modified E.coli DH5α (competent bacteria) according to standard procedures and it was purified using a NucleoBond Xtra Midi extraction kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions. Briefly, E.coli competent bacteria (they are more likely to incorporate foreign DNA as their cell walls are altered, so that DNA can pass through more easily) were thaw on ice, an then a volume of pEGFP was added and everything was incubated on ice for 30 PAMAM-based Dendrimers for Diverse Biomedical Applications 19 minutes. The mixture was heat-shocked by incubating it at 42°C for 30 seconds in a thermo-shaker TS-100 (Biosan, Latvia) and then they were placed on ice immediately (to reduce damage to the cells). Luria Broth (LB) medium was added to the cells, and incubated at 37°C for 1 hour with agitation in a thermo-shaker. Several dilutions of the transformation mixture were plated onto separate LB-amp agar plate and grown overnight at 37ºC in a bacteriological incubator (Incubat, JPSelecta, Spain). Inoculated plates were incubated inverted to prevent the condensation of water from dropping onto the agar surface, which can result in the colonies coalescing. Colonies were picked and placed in LB-kanamycin (25µg/ml), and then incubated at 37ºC in a shaking incubator KS 4000i control (IKA, Germany) overnight. The culture was transferred to centrifuge tubes and the pellet resuspended in different lysis buffers that contain Tris-EDTA, RNase, sodium hydroxide and SDS. These compounds lead to the rupture of the cell wall and membrane, and also inhibit nucleases and degrade RNA and chromosomal DNA (but not the covalently closed circular plasmid DNA). Plasmid DNA was precipitated with 60% isopropanol, washed with 70% ethanol, air-dried and finally resuspended in dH 2 O. DNA concentration and purity of pEGFP were determined by using a NanoDrop spectrophotometer (Thermo Scientific, Spain). The intactness and identity of the plasmid were confirmed by agarose gel electrophoresis. For linearization, the plasmid was digested with PStI (New England Biolabs, UK). To prepare LB-broth, the premix LB powder was dissolved (1% bacto-tryptone, 0.5% yeast extract, 1% NaCl (w/v)) (Scharlau, Scharlab, Spain) in dH 2 O while heating and then the mixture was autoclaved (20 mins, 121ºC, 2atm). The medium was kept at 4ºC. To make the LB-agar plates, the premix LB powder was dissolved (1% bacto-tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar (w/v)) (Scharlau, Scharlab, Spain) in dH 2 O while heating and then autoclave the bottle (20 mins, 121ºC, 2atm). Agar was cooled to ~55ºC and additives added when necessary, such as ampicillin (100 µg/ml) or kanamycin (25 µg/ml). A thin layer of LB Agar (~10ml) was poured into each plate and cooled until it was solid. Plates were flipped so as to avoid condensation on the agar and stored at 4ºC until usage. All the procedures were carried out at the flame (Bunsen), and all the materials (30 mins, 121ºC, 2atm) and solutions (20 mins, 121ºC, 2atm) were autoclaved or filtered (0.2 µm syringe filter) and sprayed with 70% ethanol to ensure sterile conditions so as to prevent contamination. Master in Nanostructured Materials for Nanotechnology Applications 20 3.2 Gel Retardation Assay This assay is also known as electrophoretic mobility assay and it is used to study the formation of DNA-particle complexes. The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move towards the positive pole. The rate at which the DNA will move is slowed by making the DNA move through an agarose gel. The agarose forms a porous lattice and the DNA slips through the holes. In summary, the basis of the assay is that, as the size of the DNA-particle complexes is larger than DNA alone, they should migrate more slowly than the free DNA fragments. The gel was done by mixing the agarose (Lonza, Spain) with 1X TAE (Tris-acetate- EDTA) buffer and heating until the agarose was dissolved. The agarose was cooled and ethidium bromide (a DNA intercalating agent used as a fluorescent dye) (Fermentas, Thermo Fisher Scientific, Spain) was added. The mixture was poured into a gel electrophoresis tray with a comb -to make the wells- and let dry for 45 minutes. mPEG 2000 -G3-PAMAM and pDNA were diluted in dH 2 O and Vector/pDNA complexes were prepared at various weight ratios ranging from 0.1:1 to 400:1 and incubated for 30 minutes at room temperature. The complexes were mixed with appropriate amounts of 6X loading buffer (which contains a dye to assess how fast the gel is running and a reagent to render the samples denser than the running buffer) (Takara, UK) and then electrophoresed on a 0.5% (w/v) agarose gel containing ethidium bromide (0.5 µg/ml) for 45 minutes at 100V. All the gel electrophoresis equipment used was supplied from Biorad (Spain). The location of pDNA in the gel was analyzed on a G:Box UV transilluminator (Syngene, UK). 4. EVALUATION OF BACTERIAL GROWTH 4.1 Bacterial Preparations The bacteria used in the analysis were Escherichia coli and E.coli-EGFP (Grampositive, G+), Pseudomonas aeruginosa (Gram-negative, G-), Enterobacter aerogenes(Guo Qing-Song, et al,47), Salmonella typhimurium (Guo Qing-Song, et al,47) and Staphylococus.aureus (G+) (kindly donated by the University Francisco de Vitoria, Madrid). Gram-positive bacteria retain the color of the crystal violet stain in the PAMAM-based Dendrimers for Diverse Biomedical Applications 21 Gram stain because their cell wall is composed of a thick layer of peptidologlycan; Gram-negative bacteria lose the crystal violet stain because their layer of peptidologlycan is very thin. All the microorganisms were grown at 37ºC and maintained in LB-agar plates at 4ºC; E.coli-EGFP was maintained in LB-kanamycin. Inoculated plates are incubated inverted to prevent the condensation of water from dropping onto the agar surface. To freeze the bacteria, one colony from each plate was inoculated in LB-broth and grown at 37ºC O/N while shaking. Each bacterial suspension was diluted with sterile glycerol solution to a final 15%v/v glycerol concentration. The samples were kept in ice during the process and at the end quickly transferred to a -80°C freezer for long-term storage. For thawing, bacteria were left on ice for 30 minutes and the suspension directly plated. Bacteria were incubated O/N at 37ºC. All the procedures were carried out at the flame (Bunsen), and all the materials (30 mins, 121ºC, 2atm) and solutions (20 mins, 121ºC, 2atm) were autoclaved or filtered (0.2 µm syringe filter) and sprayed with 70% ethanol to ensure sterile conditions so as to prevent contamination. 4.2 Bacterial Assays 4.2.1. LB plate assay Ampicillin (Sigma, Spain), kanamycin (Invitrogen by Life Technologies, Thermo Fisher Scientific, Spain) and mPEG 2000 -G3-PAMAM were added to LB-agar plates at different concentrations ranging from 0.5-500 µg/ml and let air-dry. Aliquots of each culture were left O/N on LB in agitation at 140 rpm and were then plated. Both antibiotics and dendrimer solutions were previously diluted in dH 2 O and filtered with a 0.2 µm syringe filter besides the flame. Plates were incubated at 37°C to allow colony formation and observed after 24h. 4.2.2. LB broth assay Ampicillin, kanamycin and mPEG 2000 -G3-PAMAM (diluted in dH 2 O and filtered with a 0.2 µm syringe filter) were added to LB-broth at 50, 100 and 500 µg/ml and then a colony from each bacteria strain was resuspended in each broth. Bacteria suspensions were checked for turbidity after 24h incubation at 37ºC while shaking at 140 rpm and the positive suspensions with the higher antibiotic or dendrimer concentration were plated in LB-agar and incubated overnight. Master in Nanostructured Materials for Nanotechnology Applications 22 4.2.3. LB microplate assay The minimum inhibitory concentration (MIC) of the PAMAM derivatives, that is, the lowest concentration of the compounds that inhibits the visible growth of P.aeruginosa and S.aureus after 18-24 h of incubation, was determined using a broth microdilution technique. A pre-inocule of P. aeruginosa and S.aureus were grown O/N in agitation in LB-broth. Serial dilutions of the bacterial suspensions were prepared in a 24-well plate (MW24) and absorbance was measured to adjust the bacteria concentration to 2x10 5 CFU/ml (Abs~0.20 at 630 nm) (stock solution). Dilutions of mPEG 2000 -G3-PAMAM were prepared in dH 2 O (50-4000 µg/ml). The stock bacterial solutions were distributed into a 96-well sterile plate (MW96) and each of the prepared dendrimer solutions was added to the wells. Control wells were composed of bacterial solutions without dendrimers (positive controls) and LB medium without bacteria but with dendrimer (negative controls). To reduce evaporation during incubation (which is critical to the assay), each of the empty outer wells of the microtiter plate was filled with sterile water to increase the water vapor pressure in the incubator. The microtiter plates were incubated at 37 °C and 140 rpm O/N. At the end of the incubation, the pellets formed were resuspended prior to scanning the optical densities using a Synergy HT (BioTek, USA) at 630 nm, which is proportional to the number of bacteria in suspension. 5. EVALUATION OF CELL GROWTH 5.1 Culture of Cell Lines Human Neuronal Gliobastoma U251MG cells (Cancer Research UK, Cell Lines Services) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/l D-Glucose, 4 mM L-Glutamine and 1.0 mM sodium pyruvate (Gibco, Invitrogen by Life Technologies, Thermo Fisher Scientific, Spain). Also, 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin (Lonza) and 1% amphotericin B (Lonza) were added to complement the media. Cells were maintained at 37ºC in normoxic conditions (21% O 2 ; 5% CO 2 ) in an Excella ECO-170 CO 2 incubator (New Brunswick Scientific, UK). When subculturing, media was removed and cells were washed with DPBS (Gibco, Invitrogen by Life Technologies, Thermo Fisher Scientific, Spain). Then, 1% trypsin solution (Trypsin-Versene 10X, Lonza) was added and cells were incubated at 37ºC for PAMAM-based Dendrimers for Diverse Biomedical Applications 23 5 minutes, allowing trypsin to break down the proteins which enable the cells to adhere to the vessel. Trypsin action was stopped by adding fresh cell culture media and a part of the cells was resuspended and dispensed into a new flask or plate. Cells were passaged every 4-5 days (80-90% confluence). Murine MSCs (bone marrow of C57BL⁄6 mouse at ≤ 8 weeks after gestation, Gibco, Invitrogen by Life Technologies, Thermo Fisher Scientific, Spain by Life Technologies, Spain) were cultured in Dulbecco’s modified Eagle’s medium (DMEM-LG) containing 1 g/l glucose, 2 mM L-glutamine and 1.0 mM sodium pyruvate (Gibco, Invitrogen by Life Technologies, Thermo Fisher Scientific, Spain). Also, 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% amphotericin B were added to complement the media. Alternatively, mMSCs were cultured in MesenCul MSC Basal Medium (Mouse), adding 10% MesenCult Mesenchymal Stem Cell Stimulatory Supplements (Mouse) (StemCell, Miltenyi Biotec, Spain), 1% penicillin/streptomycin and 1% amphotericin B. In both cases, mMSCs were maintained at 37ºC under hypoxic conditions (1% Oxygen and 5% CO 2 ) in a Forma Series II Water jacketed CO 2 Incubator, HEPA class 100 (Thermo Fisher Scientific, Spain). For subculturing, the same procedure as previously described for U251MG cells was followed, but using 2.5% trypsin (Trypsin-Versene 10X, Lonza, Spain) (as in this case cells showed a higher adherence). Cells were passaged every 6-7 days when using DMEM-LG and every 3-4 days when using MesenCult (in both cases, when cells reached 80-90% confluence). For freezing, cells were first trypsinize by using the same procedure as for passaging them; then the pellet was resuspended with freezing media (80% FBS, sterile 20% DMSO) and the cryovial (Simport, USA) was left at -80°C for a minimum of 24h; then the vial was transferred to the liquid N 2 tank for indefinite storage. When thawing, the vial was removed from the liquid N 2 tank and it was held it in the 37°C water bath until the freezing media was thawed. Gently pour cells into a falcon tube with PBS, centrifuge and resuspend in media. Media needs to be changed the next day to prevent cell damage with possible remaining DMSO. All the cultures were tested for mycoplasma and all the cell culture plastic labware was supplied by Nunc (Thermo Fisher Scientific, Spain). A vertical laminar flow hood Safemate 1.2 (BioAir, Italy) was used for all the culturing procedures. The tray was swab with 70% ethanol before and after each use Master in Nanostructured Materials for Nanotechnology Applications 24 and UV was irradiate for 10 minutes. Full protective clothing and nitrile gloves must be worn at all times when using the hood and the incubators. Cells were observed under an AE31 Research Grade Inverted Microscope (Motic, Spain) and manually counted using a Neubauer hemocytometer chamber (Blaubrand, Germany). 5.2 Viability Assays 5.2.1. Crystal Violet method Crystal violet (CV) is a triphenylmethane dye (4-[(4-dimethylaminophenyl)-phenyl- methyl]-N,N-dimethyl-aniline) that stains DNA. CV is a simple assay that is useful for obtaining quantitative information about the relative density of cells adhering to multiwell cluster dishes. Upon solubilization, the amount of dye taken-up by the monolayer and the intensity of the color produced are proportional to cell number. U251MG and mMSCs cells were seeded at a density of 2x10 3 cells/well in a MW96, or alternatively at 4x10 4 cells/well in a MW24. A stock solution of 50mg/ml was made on the moment by diluting the dendrimer in dH 2 O. Then, the solution was filtered with a 0.2µm syringe filter (25mm diameter, Nalgene, Labclinics, Spain) in the laminar flow hood and kept protected from light at 4ºC. Upon 24h incubation and without removing the media, fresh media was added to the control wells and mPEG 2000 -G3-PAMAM diluted in media at different concentrations (0.5, 1 and 5 mg/ml) was added to the rest of the wells (in triplicate). Media was removed after 24, 48 and 72h of incubation with the dendrimer and cells were fixed with 1% Glutaraldehyde for 10 mins; then, PBS was added and the plate was kept in the incubator until the day of the analysis. Then, PBS was removed and 0.1% crystal violet (dissolved in ethanol) (Roth, Spain) was added for 30 mins at room temperature. After the staining, the dye was removed washing with tap water and the plate air-dried for a couple of days at room temperature. 10% acetic acid was added to the wells to extract the dye from the cells, transferred to a new MW96 and absorbance read on a Synergy HT (BioTek, USA) plate reader at 590nm. Citotoxicity was expressed as relative viability of cells (% of the control cells incubated with medium only). All the experiments were repeated at least three times. PAMAM-based Dendrimers for Diverse Biomedical Applications 25 5.2.2. Alamar Blue method Resazurin (7-hydroxy-10-oxido-phenoxazin-10-ium-3-one) is a redox dye that is commercially available as Alamar Blue, which exhibits both colorimetric and fluorometric change that relates to cellular metabolic activity. The assay is based on the ability of viable, metabolically active cells to reduce resazurin to resorufin and dihydroresorufin. This conversion occurs intracellularly, where the oxidized form of the resazurin enters the cytosol and is converted to the reduced form 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. U251MG and mMSCs cells were seeded at a density of 1-3x10 3 cells/well in a MW96. Upon 24h incubation, media was removed and fresh media was added to the control wells and mPEG 2000 -G3-PAMAM diluted in media at different concentrations (0.5, 1 and 5 mg/ml) was added to the rest of the wells (in triplicate). The dendrimer stock solution at 50mg/ml in dH 2 O was prepared as previously described. Media was removed after 24, 48 and 72h of incubation with the dendrimer 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. The reading of the plates was done on a daily basis to prevent the cells from contaminating, so they were seeded in different plates, and each plate was discarded after measuring the fluorescence. After 3h incubation at 37ºC, fluorescence was read at 530/590 (excitation/emission) on a Synergy HT (BioTek, USA) plate reader. Citotoxicity was expressed as relative viability of cells (% compared to the control cells incubated only with medium). To set up the best conditions, different combinations were assayed (seeding concentration, culturing media, incubation time with assay reagent, etc), but in all cases the experiments were repeated at least three times. Master in Nanostructured Materials for Nanotechnology Applications 32 PART III: RESULTS AND DISCUSSION 1. STRUCTURAL ANALYSIS OF mPEG 2000 -G3-PAMAM The integrity of mPEG 2000 -G3-PAMAM was tested right after being synthesized ( 1 H MNR carried out by R. Cervera). After several weeks the integrity of the dendrimer was checked. Emergence of peaks at 1705cm-1 and 1655cm-1 shows the change in length of –C=O (stretching) due to the binding between the carboxyl of mPEG and the amide of PAMAM. 60 70 80 90 100 5001000150020002500300035004000 Wavenumbers (cm-) % T 60 70 80 90 100 5001000150020002500300035004000 Wavenumbers (cm-) % T Figure 3.1. (a) General structure of mPEG-G3-PAMAM and (b)FTIR spectrum of mPEG 2000 -G3-PAMAM dendrimer. The red arrows points at the peaks related to a change in length of –C=O. Approximate peaks at 3300cm -1 , 2800 cm -1 , 1450 cm -1 , 1300 cm -1 and 1100 cm -1 correspond to H-O, C-H, N-H (amide II), C-O and C-O-C, respectively. a b PAMAM-based Dendrimers for Diverse Biomedical Applications 33 As happens in higher dendrimer generation (in this case a lower generation but completely covered with long PEG chains), the spectral lines became weak and, as a result, we did not obtain a line structure, but a general molecular weight distribution can be observed (Fig.3.2). From that spectrum we calculate a theoretical mass of ~ 70000 Da. Figure 3.2. MALDI-LP spectrum of mPEG 2000 -G3- PAMAM using DHB as a matrix. The red arrow points at the calculated mass. Figure 3.3 . 1 H NMR spectrum of mPEG 2000 - G3 - PAMAM in DMSO - d6. Red arrows point to CH 2 -PEG, CH 3 -PEG and DMSO (from left to right); red box locates PAMAM-dendrimer-CH 2 . Master in Nanostructured Materials for Nanotechnology Applications 34 The results suggested that a mPEG-G3-PAMAM dendrimer complex was formed. All the data presented here agrees with previous published results (Kim, et al,48). Finally, the integrity of mPEG 2000 -G3-PAMAM was established by 1 H NMR after 18 weeks up to 8 months of keeping it in darkness and room temperature. The dendrimer was further dissolved in deuterated water and kept protected from light, exposed to light at 4ºC, room temperature and 37ºC for periods 72h. Any of the spectra show differences compared to the one obtained before (data not shown) concluding that the dendrimer is stable at room temperature for long periods of time. 2. DNA-DENDRIMER COMPLEX FORMATION Currently the practical use of unmodified PAMAM dendrimers has been questioned by reports that show relatively high citotoxicity as a result of their high surface charge density. Pegylation increases bioavailability of polymer-DNA complex by enhancing solubility and increasing the circulation time in the bloodstream; also facilitates dissociation of DNA inside the cell (Fant, et al,28). Before trying the transfection efficiency of the dendrimer, the amount of plasmid DNA that binds the dendrimer is optimized so neither vector nor therapeutic agent is wasted. 1:400 1:200 1:100 1:40 1:20 1:13 1:10 Ctr- 5000pb 1000pb 1:400 1:200 1:100 1:40 1:20 1:13 1:10 Ctr- 5000pb 1000pb 1:400 1:200 1:100 1:40 1:20 1:13 1:10 Ctr- 5000pb 1000pb Figure 3.4 . Gel Retardation Assay. Ratio Plasmid:Dendrimer (increasing amounts of plasmid). Lane 1: DNA marker (with known bands for estimating the molecular weight of the sample bands). Lane 2: negative control with dendrimer alone. Lanes 3-9: mixtures of pEGFP (concentrations of pEGFP ranging from 0.125-5 µg) and dendrimer (50 µg). There is no retention of Dendrimer-Plasmid complexes in the wells (red box). The intensity of the bands related to pEGFP increases with increasing plasmid concentration (yellow circle). PAMAM-based Dendrimers for Diverse Biomedical Applications 35 When incubation of the dendrimer with the plasmid DNA, if the ratio is optimal, the vector incorporates all the plasmid and the complex formed is retained in the well. When the ratio dendrimer:plasmid is higher than the optimal one, extra-not bound plasmid runs along the gel. The gel retardation assay demonstrated that DNA would not package mPEG 2000 -G3- PAMAM dendrimer at D/P ratios up to 400 (Fig. 1). Because it is completely covered with mPEG, G3-PAMAM has no external positive charge to bind the plasmid DNA (negatively charged) and then it will not be useful for plasmid transfections into the cells. 3. EVALUATION OF BACTERIAL GROWTH We have evaluated the antibacterial activity of mPEG 2000 -G3-PAMAM against E.coli and E.coli-EGFP (Gram-positive, G+), P. aeruginosa (Gram-negative, G-), E. aerogenes(Guo Qing-Song, et al,47), S. typhimurium (Guo Qing-Song, et al,47) and S. aureus (G+). The cytoplasmatic cell membrane is the main target for many antibacterial agents, and PAMAM dendrimers have shown great potential interacting and disrupting them (Cheng, et al,37). In a first attempt, bacteria were incubated in plates containing LB agar with a dilution of the dendrimer at a specific concentration. In order to compare the activity of the dendrimer with effective antibiotics commonly used in the lab, similar concentrations were tested (from 0.5-100 µg/ml). As it is shown in Figure 3.5, grow inhibition is observed in Ampicilin and Kanamycin plates (used as positive controls), but no effect can be observed for any of the bacteria with the dendrimer (plates with antibiotics and dendrimer at lower concentrations are not shown). In a second attempt, bacteria were incubated with the dendrimer in solution, so as to favor interactions between both. Again, all the bacteria were found to be resistant to the dendrimer at concentrations of 100-500 µg/ml (Fig. 3.6) (lower dendrimer concentrations are not shown). Master in Nanostructured Materials for Nanotechnology Applications 36 Eventually, a more accurate and reproducible assay was used to asses the activity of the dendrimer. In this case, instead of incubating the dendrimer with an unknown number of microorganisms, we adjusted the number of bacteria to a certain value of cfu/ml prior to incubation with mPEG 2000 -G3PAMAM. Then, the optical density at 635 nm was measured, which is proportional to the number of bacteria. In this case we only used P. aeruginosa and S. aureus, since both are common pathogens in ocular and vaginal infections and have been previously tested with PAMAM-derivates successfully (Lopez, et al,33). Figure 3.6. Antimicrobial activity of mPEG2000-G3-PAMAM using a broth assay. All the bacteria shown here were incubated with 500 µg/ml. LB alone was used as the negative control (no turbidity). The increased turbidity is a sign of bacterial growth. Figure 3.5. Antimicrobial activity using a plate assay using (a) ampicilin (100 µg/ml), (b) kanamycin (25 µg/ml) and (c) mPEG2000-G3-PAMAM (100 µg/ml). a b c PAMAM-based Dendrimers for Diverse Biomedical Applications 37 As expected, mPEG2000-G3-PAMAM is more efficient in inhibiting the growth of P.aeruginosa (Gram-) (concentrations of 100 µg/ml and above) compared to S.aureus (Gram+). This can be explained taking into account that the antibacterial action of the dendrimers involves disruption of the cytoplasmic membrane of the bacteria, and in the case of Grambacteria, the top of the plasma membrane is covered by negativelycharged proteins that facilitate membrane permeation and hence killing of the bacteria. We will check in the near future another 2 Gram – bacterias, E. aerogenes (Guo Qing- Song, et al,47) and S. typhimurium (Guo Qing-Song, et al,47) to test if is a general effect in all the Gram - bacterias. In the initial assays the bacteria appear to be resistant to the dendrimer at concentrations up to 500 µg/ml (Fig. 3.7). In that case we looked at the turbidity to determine the result, and in this case, although we measure the OD to obtain more specific data, we could also see growth of the bacteria in all the wells, meaning that the bacteriostatic effects could be identical in both assays but we did not have enough resolution. It is also possible that the character of the PEG chains to some extent enhances the permeation through the cell membrane. Moreover, the fact that the dendrimer retains antibacterial activity at high concentrations (4 mg/ml) could be an effect of the detachment of some of the PEG chains from the PAMAM and the dendrimer. Higher Figure 3.7. Antimicrobial activity of mPEG 2000 -G3-PAMAM using a modified broth microdilution assay. Gram+ bacteria (blue) and Gram- (pink). 0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 0 100 200 500 1000 2000 4000 [mPEG 2000 -G3-PAMAM] (µg/ml) [OD] 630 S.aureus P.aeruginosa Master in Nanostructured Materials for Nanotechnology Applications 38 concentrations of dendrimer could be tested, but they would be substantially higher than those of common antibiotics, and in general terms, more expensive, and then some of the advantages would be lost. Although PAMAM dendrimers strongly bind to bacteria and third generation has proved to be more effective because they easily penetrate towards the bacterial membrane (Lopez, et al,33), we can conclude that complete PEGylation deactivates the dendrimer against bacteria but our dendrimer still retains some of the antibacterial effect against Gram - bacterias. 4. EVALUATION OF CELLULAR GROWTH AND ALTERATIONS The citotoxicity of PAMAM dendrimers in mammalian cells lines due to the nature of the surface charge has been noted. Before using this type of vectors in any in vivo application, a citotoxicity profile must be done in vitro. In our study, the citotoxicity of mPEG 2000 -G3-PAMAM dendrimer was evaluated using four commonly used methods: Crystal Violet (CV), Alamar Blue (AB), MTT and Trypan Blue (TB). At least, two different cell types must be tested, our cells of interest (MSCs) and a tumoral cell line (U251MG). An interesting phenomenon was observed when measuring the cell viability of both U251MG (Fig. 3.8a) and mMSCs (Fig. 3.8b) cells after incubation with increasing concentrations of the dendrimer at different times (Hoskins, et al,49). The presence of the dendrimer resulted in greater absorption readings compared to the control. This phenomenon could be explained by the adherence of the sticky polymers to the well surface. The value of cell viability that appeared was still larger compared to the visual inspection of viable cells under microscope (Fig. 3.9A, 3.9B). Until 42h, cells show more proliferation at lower dendrimer concentrations; at 72h and concentrations between 1-5 mg/ml, the dendrimer shows citotoxicity effects. PAMAM-based Dendrimers for Diverse Biomedical Applications 39 Figure 3.8. Cell Viability assessed by the four methods. ( a) U251MG, (b) mMSCs. Ctr, 0.5 mg/ml, 1 mg/ml, 5 mg/ml 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability Crystal Violet Alamar Blue MTT Trypan Blue 0 20 40 60 80 100 120 140 160 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability Crystal Violet Alamar Blue MTT Trypan Blue 0 20 40 60 80 100 120 140 160 24 48 72 Incubation Time (h) % Cell Viability a Crystal Violet Alamar Blue MTT Trypan Blue 0 20 40 60 80 100 120 140 160 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability Crystal Violet Alamar Blue MTT Trypan Blue 0 20 40 60 80 100 120 140 160 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability 0 20 40 60 80 100 120 140 160 180 200 24 48 72 Incubation Time (h) % Cell Viability b Master in Nanostructured Materials for Nanotechnology Applications 40 Based on the above observations, trypan blue exclusion was used as the standard method to validate the cell viability data obtained by the above assays (Fig. 1 and 1). This method involved direct counting of viable cells and hence eliminated the possibility of interference from occurring. The results demonstrated a large difference between the cell viability data from trypan blue counting and all other three enzyme activity-based assays. These values correlated well with visual estimations when observing under the microscope. Figure 3.9. Inverted microscope images (4X) of both cell lines after 72h incubation with and without the dendrimer. A) U251MG (a, control with no dendrimer; b, 0.5 mg/ml; c, 1 mg/ml; d, 5 mg/ml); B) mMSCs (a, control with no dendrimer; b, 0.5 mg/ml; c, 1 mg/ml; d, 5 mg/ml). Red arrows indicate the presence of cell accumulations (mMSCs-a) or holes in the monolayer. A b c d ab c d a a b d c a b d c B PAMAM-based Dendrimers for Diverse Biomedical Applications 41 Images of both cell lines at 72h of incubation show less cellular growth with higher dendrimer concentrations (Fig. 3.9Ac, 3.9Ad, 3.9Bc, 3.9Bd). In the case of mMSCs, cells that were incubated only with medium without dendrimer tend to form agglomerations (Fig. 3.9Ba), which is a sign of uncontrolled growth that ends in the cells differentiation and so, of their lost in multipotency. In order to measure the level of interference with CV, AB and MTT, the same assays were carried out, but incubating the cells with dendrimer and without reagents. Furthermore, dendrimer with assay reagents in the absence of cells were also analyze. No significant effect on absorbance or fluorescence readout was observed (data not shown). Therefore, the increased absorbance and fluorescence could appear due to a combination of cells, dendrimer and assay reagents. Also, the changes in the metabolic state of the cells could be implicated. There is evidence that for the AB assay, there is accumulation of colorless products with time of incubation, which can also affect the final results. To analyze whether culturing the cells with a different formulation of the dendrimer worked better in terms of citotoxicity and rate of proliferation, the dendrimer was incubated in gelatin in the bottom of the wells prior to seeding them, so they could grow on top of the monolayer gelatin-dendrimer. This way the dendrimer was intended to be more accessible, or for longer times to the cells, as it would be trapped in the gelatin. 0 20 40 60 80 100 120 140 160 24 48 72 96 Incubation Time (h) % Cell Viability Ctr 0'5mg/mL 1mg/mL 5mg/mL 0 20 40 60 80 100 120 140 160 24 48 72 96 Incubation Time (h) % Cell Viability Ctr 0'5mg/mL 1mg/mL 5mg/mL 0 20 40 60 80 100 120 140 160 24 48 72 96 Incubation Time (h) % Cell Viability Ctr 0'5mg/mL 1mg/mL 5mg/mL 0 20 40 60 80 100 120 140 160 24 48 72 96 Incubation Time (h) % Cell Viability Ctr 0'5mg/mL 1mg/mL 5mg/mL Figure 3.10. Cell Viability by AB, culturing over a monolayer of dendrimergelatin. On top, U251MG cells; on the bottom, mMSCs. 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