PNIPAM And NIPAM BAM CO Polymer Nanoparticles Biology Essay

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Poly N-isopropylacrylamide (PNIPAM) and N-isopropylacrylamide-co-N-tert-butylacrylamide (NIPAM/BAM) copolymer particles with three different ratios of the comonomers (85:15, 65:35, and 50:50 NIPAM/BAM) were made available by University College Dublin through the "Integrated NanoScience Platform for Ireland" collaborative programme (www.inspirenano.com). They were synthesised by free radical polymerisation. The procedure for the synthesis was as follows: 2.8g monomers (in the appropriate wt/wt ratio), and 0.28g crosslinker (N,N-methylenebisacrylamide) was dissolved in 190 ml MilliQ water (MQ) with 0.8 g Sodium Dodecyl Sulphate (SDS) and degassed by bubbling with nitrogen gas for 30 minutes. Polymerisation was induced by adding 0.095g ammonium persulfate initiator in 10 ml MQ water and heating at 70°C for 4 hours. Particles were extensively dialysed against MQ water for several weeks, changing the water daily, until no traces of monomers, crosslinker, initiator or SDS could be detected by proton NMR (spectra acquired in D2O using a 500 MHz Varian Inova spectrometer). Particles were freeze-dried and stored in the fridge until used. Due to the inverse solubility of PNIPAM and NIPAM/BAM particles, solutions were prepared by dispersing the particles on ice to ensure good solubility of the particles (i.e. to ensure that the solutions are below the lower critical solution temperature of the particles and thus that polymer-water contacts are more favourable than polymer-polymer contacts which would result in uptake of water and swelling of the particles), before gradually warming them to the test conditions.

Fluorescently tagged NIPAM nanoparticles with nominally 500 fluorescent labels per particle were also synthesized within the "Integrated NanoScience Platform for Ireland" collaborative programme (www.inspirenano.com). In brief, 0.1 g of SDS was mixed with 0.0044 g of methacryloxyethyl thiocarbamoyl Rhodamine B in 10 ml of MQ water and then sonicated using a Covaris S2 system at a frequency of 450 kHz for 500 seconds until most of the dye was visibly dissolved. The solution was transferred into a falcon tube adding an additional 10 mL MQ water together with the rest the SDS (0.3 g) and then sonicated using an ultrasonic bath (Branson 1510) at a frequency of 42 kHz for 5 hours continuously until the dye was completely dissolved in the SDS. The monomers (1.4 g of NIPAM, 0.14 g of cross linker) were added to this solution with 75 ml of MQ water, stirred for 30 minutes under nitrogen flow to remove dissolved O2, heated at 70°C and then the synthesis was performed by adding a degassed solution composed of 0.0475 g of initiator diluted in 5 ml of MQ water. The reaction was carried out for 12 hours at 70°C and under nitrogen flow. The labelled particles were dialysed against ethanol for 6 days and then extensively dialysed in ultrapure water, freeze dried and stored at 4°C.

2.1.2 PAMAM (Poly amidoamine) dendrimers

G-6

G-5

G-4

Figure 2.1 : PAMAM dendrimer G-4 , G-5 and G-6

Polyamidoamine (PAMAM) dendrimers G-4, G-5 and G-6 (Figure 2.1) having an ethylenediamime core (StarburstTM, Dendritech Inc.) were purchased from Sigma-Aldrich (Ireland). The average molecular weight of G-4, G-5 and G-6 is 14,215, 28,825 and 58,048 and they contain 64, 128 and 256 surface amino groups respectively (www.dendritech.com).

2.2 Experimental protocol

2.2.1 Particle Characterisation

2.2.1.1 Particle size measurement

Dynamic Light Scattering is the measurement of the size and size distribution of particles emulsions and molecules dispersed or dissolved in a liquid. Particles, emulsions and molecules in suspension undergo Brownian motion. This is the motion induced by the bombardment by solvent molecules that themselves are moving due to their thermal energy. If the particles or molecules are illuminated with a laser, the intensity of the scattered light fluctuates at a rate that is dependent upon the size of the particles as smaller particles are "kicked" further by the solvent molecules and move more rapidly. Analysis of these intensity fluctuations yields the velocity of the Brownian motion and hence the particle size using the Stokes-Einstein relationship. The diameter that is measured in Dynamic Light Scattering is called the hydrodynamic diameter and refers to how a particle diffuses within a fluid. The basic construction of the dynamic light scattering instrument is shown in figure 2.2

Figure 2.2. Schematic diagram of a conventional dynamic light scattering instrument. (http://www.malvern.com/LabEng/technology/dynamic_light_scattering/classical_90_degree_scattering.htm).

The particle size distributions of PNIPAM and NIPAM/BAM copolymer nanoparticles in the appropriate assay media were analyzed using a Zeta sizer (Malvern Instruments, UK). For a typical experiment, approximately 1000 µg/ml concentration suspension of nanoparticles in MQ water and the respective assay media (i.e. algal medium [AM], Daphnia medium [DM] and Microtox® diluent [MD]) were analysed as a function of temperature from 0oC to 30oC with an interval of 5oC due to the thermoresponsive nature of these particles. The hydrodynamic diameter of PNIPAM nanoparticles was measured in the cell culture media as a function of temperature from 30 to 38 oC, as the mammalian cells are grown at 37 oC, to understand the behaviour of these particles in the appropriate experimental conditions. In the case of PAMAM dendrimers, approximately 20 M concentration suspensions of dendrimer nanoparticles in the respective assay media DM, MD and Thamnocephalus medium (TM), and the cell culture medium, Dulbecco's Modified Medium Nutrient Mixture/F-12 Ham [DMEM], with 5% foetal calf serum (FCS) supplement (PLHC-1) and 10% serum supplement (RTG-2) were analysed at 20oC. The pH of the various test media, before and after exposure to G-4, G-5 and G-6 dendrimers was measured using a HQ11d Single-Input pH meter (Hach Company, Colorado).

2.2.1.2 Zeta potential measurement

Sometimes thought of as a 'charge' measurement, measurement of zeta potential is used to assess the charge stability of a disperse system, and assist in the formulation of stable products. Zeta potential may be related to the surface charge in a simple system, but equally well may not. The zeta potential can even be of opposite charge sign to the surface charge. One of the most important lessons is that it is the zeta potential that controls charge interactions, not the charge at the surface. It is one of the main forces that mediate inter-particle interactions. Particles with a high zeta potential of the same charge sign, either positive or negative, will repel each other. Conventionally a high zeta potential can be high in a positive or negative sense, i.e. <-30mV and >+30mV would both be considered as high zeta potentials. For molecules and particles that are small enough, and of low enough density to remain in suspension, a high zeta potential will confer stability, i.e. the solution or dispersion will resist aggregation.

Zeta potential is measured by applying an electric field across the dispersion. Particles within the dispersion with a zeta potential will migrate toward the electrode of opposite charge with a velocity proportional to the magnitude of the zeta potential.

A

B

Figure. 2.3 A. Zeta sizer (Malvern Instruments) and B. Schematic representation of zeta potential.(http://www.malvern.com/LabEng/technology/zeta_potential/zeta_potential_LDE.htm)

The zeta potential of PNIPAM and NIPAM/BAM nanoparticles and PAMAM dendrimers was measured in the respective assay media using a Zeta sizer (Malvern Instruments, UK, Figure 2.3A). The zeta potential measurements of PNIPAM and NIPAM/BAM copolymer nanoparticles were conducted at 20 oC, using a concentration of 1000 µg/ml. In the case of PAMAM dendrimers, measurements were conducted at 20oC, using a 20 M concentration.

2.2.1.3 Spectroscopic analysis

As it has previously been demonstrated that some nanoparticles can interact and bind with various molecular constituents of cell culture media (Casey et al., 2008), absorption spectroscopic analysis of each dendrimer in the different cell culture media (DMEM, RTG-2 and PLHC-1) was performed using a Perkin Elmer Lambda 900 UV/visible/NIR absorption spectrometer. Changes to the spectroscopic profile of the medium can result from changes to the effective composition of the medium due to molecular adsorption to the particles. This may lead to secondary toxic effects due to medium depletion (Casey et al., 2008).

In the case of NIPAM/BAM 65:35 and NIPAM/BAM 50:50, large aggregates are formed, due to the low LCST, which leads to both materials floating in the cell culture media so these two particles were deemed unsuitable for cytotoxicity assessment. However, PNIPAM and NIPAM/BAM 85:15 particles were found to be nontoxic to the fish cells. Therefore the indirect toxicity by PNIPAM and NIPAM/BAM copolymer particles, due to medium depletion effect was not analysed.

2.2.1.4 Surface Area measurement

BET theory governs the physical adsorption of gas molecules on a solid surface and serves as the basis for an important analysis technique for the measurement of the specific surface area of a material. In 1938, Stephen Brunauer, Paul Hugh Emmett, and Edward Teller published an article about the BET theory (Brunauer et al., 1938) for the first time; "BET" consists of the first initials of their family names. The concept of the theory is an extension of the Langmuir theory, which is a theory for monolayer molecular adsorption, to multilayer adsorption with the following hypotheses: (a) gas molecules physically adsorb on a solid in layers infinitely; (b) there is no interaction between each adsorption layer; and (c) the Langmuir theory can be applied to each layer.

Figure 2.4. Gemini series surface area analyser (Micromeritics, USA)

BET surface area measurements of PNIPAM and NIPAM/BAM co-polymer nanoparticles were performed using a Gemini series surface area analyser (Micromeritics, USA) (Figure 2.4). For the experiment, approximately 0.5 g particles of each of the materials were degassed with nitrogen gas at a constant temperature of 25 oC for two hours prior to surface area measurements being recorded. However, in the case of the PAMAM dendrimers it was not possible to measure BET surface area because the dendrimers are supplied by the manufacturer in methanol (in suspension form) and for BET surface area measurement, powdered samples are required.

2.2.1.5 TEM (Transmission electron microscopy) study

In the case of PNIPAM particles, particle size was also determined by Electron Microscopy. Samples were prepared by negative-contrast staining as described previously (Gorelov et al 1997). Briefly, stock solutions of tungstophosphoric acid (TPA, 200 mg / ml) (Fluka) and labelled NIPAM nanoparticles (5mg / ml) were prepared in water, and were left in a drying cabinet for about 2 hours at 55°C. The mixing of the final solution and the sample preparation was performed in the drying cabinet at a constant temperature of 55 °C. The final solution contained 20 mg / ml of TPA and 4.5 mg /ml of NIPAM nanoparticles, and was left in the drying cabinet for about 15 minutes together with the TEM grids. A drop of this final solution was placed on the grid and immediately soaked with filter paper in order to leave on the grid a thin film of nanoparticles, in this way minimising the nanoparticle aggregation during the drop drying time. Samples were investigated in a TECNAI G 2 12 TWIN TEM using an acceleration voltage of 120 kV and objective aperture of 20 μm. Digital images were recorded with a MegaView III (SIS) camera.

2.2.2 Ecotoxicity tests

Each ecotoxicity test was performed in two stages. A preliminary or range finding test was conducted which determined the range of concentrations of interest for the definitive test. The definitive test used a concentration range (at least five concentrations) in which effects were likely to occur, thereby permitting the calculation of the respective Effective Concentrations (EC50) or Lethal Concentrations (LC50), No Observed Effect Concentration (NOEC), and Lowest Observed Effect Concentration (LOEC). The acute toxicity of each dendrimer was investigated in the four test systems representing different trophic levels. The cytotoxicity of the dendrimers was also evaluated in two fish cell lines, RTG-2 and PLHC-1, to represent vertebrate species. The details of each of the cell lines are given in sections 2.2.2.5.1 and 2.2.2.5.2, respectively.

2.2.2.1 Microtox® test

The acute toxicity of each dendrimer and NIPAM/BAM series of nanoparticles to the marine bacterium Vibrio fischeri was determined using the 90% basic test for aqueous extract protocol (Azur Environmental, 1998). Lyophilised Vibrio fischeri bacteria (NRRL B-11177) and all Microtox® reagents were obtained from SDI Europe, Hampshire, UK. Phenol was used as a reference chemical and a basic test for phenol was run for every fresh vial of bacteria to ensure the validity of all tests. Readings of bioluminescent response were measured using a Microtox® Model 500 analyser (Figure 2.4) and the acute toxicity data was obtained and analysed using the Microtox Omni software (SDI Europe, Hampshire, UK). Five, fifteen and thirty minute EC50 tests were performed.

Microtox® Model 500 analyser Morphology of Vibrio fischeri

Figure 4. Microtox® Model 500 analyser and the Morphology of Vibrio fischeri (http://www.google.ie/images).

2.2.2.2 Microalgae growth inhibition assay

Assessment of the acute toxicity of the materials to the freshwater algae Pseudokirchneriella subcapitata (Figure 2.5) was conducted in accordance with OECD Guideline 201 (2002). Pseudokirchneriella subcapitata CCAP 278/4 was obtained from the Culture Collection of Algae and Protozoa (CCAP) Argyll, Scotland. All microalgae growth inhibition tests were conducted at 20 ± 1oC with continuous shaking at 100 rpm and continuous illumination of 10,000 lx. The initial algal density of all flasks was 1x104 cell ml-1 in a final volume of 20 ml. Negative controls were incorporated for each test containing only algal growth media and algal inoculum. The cell density of each replicate was measured after 72 h using a Neubauer Improved (Bright-Line) chamber (Brand, Germany). Average specific growth rate () and percentage inhibition of average specific growth rate (%Ir) relative to controls were calculated for each concentration. The reference chemical potassium dichromate was employed as a positive control to ensure validity of the test method.

Figure 2.5. Pseudokirchneriella subcapitata. (http://www.google.ie/images)

2.2.2.3 Thamnotoxkit FTM

The acute toxicity of the materials was also evaluated using the freshwater shrimp Thamnocephalus platyurus (Figure 2.6.). This toxicity test was purchased in kit form from SDI Europe (Hampshire, UK) and the test was performed according to manufacturer's instructions (Thamnotoxkit, Fâ„¢. 1995). Briefly, the test is a 24 h LC50 bioassay, which is performed in a 24-well test plate using instars II-III larvae of the shrimp, which are hatched from cysts. Hatching was initiated 24 h prior to the start of the test. Upon hatching, shrimp were exposed to various concentrations of each dendrimer and were incubated at 25oC for 24 h in the dark. The test endpoint was mortality. The number of dead shrimp for each concentration was recorded and the respective LC50 was determined. Potassium dichromate was used as a positive control.

Figure 2.6. Thamnocephalus platyurus . (http://www.google.ie/images)

2.2.2.4 Daphnia magna acute immobilisation assay

Acute toxicity immobilization tests were performed on each of the dendrimers according to the British standard (BS EN ISO 6341, 1996). Daphnia magna (Figure 2.7) were originally obtained from TNO laboratories (the Netherlands) and were cultured in static conditions at 20 ± 1oC over a 16 h/8 h light/dark photoperiod. Daphnid sensitivity was verified by determining the 24 h EC50 using potassium dichromate. Acute toxicity tests were performed on Daphnia magna neonates that were less than 24 h old. Four replicates were tested for each test concentration and five neonates were used in each replicate. There was no feeding during the tests. Immobilisation (no independent movement after gentle agitation of the test liquid for 15 seconds) was determined visually after 24 and 48 h exposure to each dendrimer nanoparticle.

Figure 2.7 . Daphnia magna. (http://www.google.ie/images)

2.2.2.5 Cell culture

2.2.2.5.1 RTG-2, rainbow trout gonadal cells

RTG-2, rainbow trout gonadal cells (Catalogue no. 90102529) (Figure 2.8a) were obtained from the European Collection of Cell Cultures (Salisbury, UK). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 45 IU ml-1 penicillin and 45 µg ml-1 streptomycin. The RTG-2 medium was also supplemented with 25 mM HEPES and 1% non-essential amino acids. Cultures were maintained in a refrigerated incubator (Leec, Nottingham, UK) at a temperature of either 20°C under normoxic atmosphere. For subculture the cells were detached using Versene/trypsin solution (1 mM EDTA/0.25 % trypsin) in Ca2+ and Mg2+ free Hanks Balanced Salts Solution (HBSS).

2.2.2.5.2 PLHC-1 cells

PLHC-1 cells (CRL-2406) (Figure 2.8b) were derived from a hepatocellular carcinoma in an adult female topminnow (Poeciliopsis lucida) and were obtained from the American Type Culture Collection. Cells were maintained in DMEM supplemented with 5 % FCS, 45 IU ml-1 penicillin, and 45 µg ml-1 streptomycin. Cultures were maintained in a refrigerated incubator (Leec, Nottingham, UK) at a temperature of 30°C under normoxic atmosphere. For subculture the cells were detached using Versine/trypsin solution (1 mM EDTA/0.25 % trypsin) in Ca2+ and Mg2+ free Hanks Balanced Salts Solution (HBSS).

2.2.2.5.3 SW4 80 cells

SW480 cells (ATCC, CCL-228) (Figure 2.8c) a primary adenocarcinoma cell line of the colon, were cultured in DMEM F-12 HAM with 2mM L-glutamine supplemented with 10% FCS, 45 IU ml-1 penicillin and 45 IU ml-1 streptomycin at 37°C in 5% CO2 . For subculture, the cells were detached using Versene/trypsin solution (1 mM EDTA/0.25 % trypsin) in Ca2+ and Mg2+ free Hanks Balanced Salts Solution (HBSS).

2.2.2.5.4 HaCaT cells

HaCaT cells, an immortal non-cancerous human keratinocyte cell line (Figure 2.8d) (Kindly provided by Prof. Dr. Boukamp, Heidelberg), were also cultured in DMEM F-12 HAM with the addition of 1µg/ml hydrocortisone (Smola et al., 1993). For subculture, the cells were detached using Versene/trypsin solution (1 mM EDTA/0.25 % trypsin) in Ca2+ and Mg2+ free Hanks Balanced Salts Solution (HBSS).

2.2.2.5.5 J774A.1 cells

J774A.1 is a mouse macrophage cell line, (ECACC, 91051511) (Figure 2.8e) derived from a tumour in a female BALB/c mouse. J774A.1 cells were cultured in DMEM with 2mM L-glutamine supplemented with 10% FCS, 45 IU ml-1 penicillin and 45 IU ml-1 streptomycin at 37°C in 5% CO2 . For subculture, the cells were detached using Versene/trypsin solution (1 mM EDTA/0.25 % trypsin) in Ca2+ and Mg2+ free HBSS.

A

C

B

D

E

Figure 2.8. Morphology of, A. RTG-2 cells, B. PLHC-1 cells, C. HaCaT cells. D. SW 480 cells, E. J774A.1 cells.

2.2.2.6 Cytotoxicity assays.

For cytotoxicity tests, with the RTG-2 cells, 96 well plates were seeded with 100 µl of the following cell suspension concentrations: 2 x 105 cells per ml for 24 h exposure periods, 1.8 x 105 cells per ml for the 48 h exposures, and 1.6 x 105 cells per ml for the 72 and 96 h exposure periods (Davoren and Fogarty 2006). For PLHC-1 cell exposures, 100 µl of the following cell suspension concentrations: 8 x 105 cells per ml for 24 h, 6 x 105 cells per ml for 48 h, 4 x 105 cells per ml for 72 and 2 x 105 cells per ml for the 96 h exposure. For the case of HaCaT and SW 480, cells are plated at a seeding density of 1 x 105 cells/ml for the 24 hour test, 6 x 104 cells/ml for the 48 hour test, 4 x 104 cells/ml for the 72 hour and 2 x 104 cells/ml for the 96 hours in 96 well plates. For the J774A.1 tests, cells were plated at a seeding density of 1 x 105 cells/ml for 24 hour exposure experiments. The plates were kept in a CO2 incubator for 24 hours for proper attachment of cells on the surface of the 96 well plates.

Test particles were prepared in a reduced serum medium (5% FCS) to minimise the effects of protein binding by the particles. For the cytotoxicity of all test particles, a range of concentrations of nanoparticles was tested to establish a preliminary range finding tests (within 10 to 90 % cytotoxic response) with each cell line. Six replicate wells were used for each control and test concentration per microplate. After each incubation period (24, 48, 72, or 96 h), the test medium was removed; cell monolayers washed with phosphate buffered saline (PBS) and prepared for the cytotoxicity assays. In the case of NIPAM/BAM copolymer nanoparticles, the cytotoxicity of PNIPAM and NIPAM/BAM 85:15 was studied in RTG-2 cells. NIPAM/BAM 65:35 and NIPAM/BAM 50:50 were demonstrated to form large aggregates at this temperature, due to the low LCST, which led to both materials floating in the cell culture media. For a cytotoxicity assessment the particles should be fully dispersed and capable of interaction with the cells so in this case it was not considered practical to test these particles with the cell line.

2.2.2.6.1 Alamar blue (AB) assay

Alamar blue (AB), a water-soluble dye that has been previously used for quantifying in vitro viability of various cells (Fields and Lancaster, 1993; Ahmed et al., 1994). Alamar Blue (AB) uptake was used as a cytotoxicity assay. The assay was carried out according to the manufacturer's instructions. Briefly, control media or test exposures were removed; the cells were rinsed once with PBS and 100µl of AB medium (5% v/v solution of AB) prepared in fresh media (without FCS or supplements) were added to each well. When the AB dye was added to cell cultures, the oxidized form of the AB enters the cytosol and is converted to the reduced form by mitochondrial enzyme activity, accepting electrons from NADPH, FADH, FMNH, and NADH as well as from the cytochromes. This redox reaction is accompanied by a shift in colour from indigo blue to fluorescent pink, which can be easily measured by colorimetric or fluorometric analysis (Al-Nasiry et al., 2007). After 3 h of incubation, AB fluorescence was measured at the excitation and emission wavelengths of 540 nm and 595 nm respectively, in a microplate reader (TECAN GENios, Grodig, Austria). The percentage of cell viability was determined by comparison with cells which were not exposed to nanoparticles i.e. the control group.

2.2.2.6.2 MTT Assay

This is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. A parallel set of plates was set up for the MTT assay and seeded and exposed in an identical manner to that described in AB assay. After desired exposure time points to nanoparticles, the control medium or test exposures was removed, the cells were washed with PBS and 100 µl of freshly prepared MTT in media (0.5 mg/ml of MTT in un-supplemented media) were added to each well. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. After 3 h incubation, the medium was discarded and the cells were rinsed with PBS and 100 µl of DMSO were added to each well to extract the dye. The plates were shaken at 240 rpm for 10 min and the absorbance was measured at 595 nm in a microplate reader (TECAN GENios, Grodig, Austria). Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells.

2.2.2.7 Internalisation study of fluorescently labelled PNIPAM nanoparticles

Methacryloxyethyl thiocarbamoyl rhodamine B labelled PNIPAM nanoparticles were used for the uptake study in the HaCaT and SW480 cells. HaCaT and SW480 cells were seeded at a density of 25,000 in glass bottom petri dishes. The petri dishes were kept in a CO2 incubator at 37 oC for 24 h. After attachment, the cells were exposed to different concentrations of fluorescent nanoparticles and after a 24 hour exposure the monolayer of cells was washed with PBS to remove external particles. The particles in the cells were visualised by excitation at 543 nm and fluorescence emission was collected above 560 nm using a confocal laser scanning microscope (LSM 510 META, Zeiss, Germany, Figure 2.9). Fluorescence and phase contrast images were recorded from a minimum of 3 areas per sample.

Figure 2.9 Confocal laser scanning microscope (Zeisse LSM 510 META).

2.2.2.8 Co-localisation study of the fluorescently labelled PNIPAM nanoparticles

Co-localisation studies of the labelled PNIPAM nanoparticles were performed in the HaCaT cells using lysotracker green (http://probes.invitrogen.com). HaCaT cells were seeded at a density of 25,000 in glass bottom petri dishes. The petri dishes were kept in a CO2 incubator at 37 oC for 24 h to attach the cells on the glass surface. After attachment, the cells were exposed to different concentrations (30 and 50, mg/l) of fluorescent nanoparticles and after 24 hour exposure the monolayer of cells was washed with PBS. The cells were then incubated for 30 minutes with 75nM concentration of lysotracker in a CO2 incubator at 37 oC. The particles in the cells were visualised using excitation at 543 nm and fluorescence emission was collected above 560 nm, whereas fluorescence from lysosomes was recorded using 488 nm excitation, emission being measured through a 505-530 nm bandpass filter in both cases using a confocal microscope (LSM 510 META, Zeiss, Germany). Fluorescence and phase contrast images were recorded from a minimum of 3 areas per sample.

2.2.2.9 Intracellular Reactive Oxygen Species (ROS)

Intracellular reactive oxygen species were measured by a fluorimetric assay using Carboxy H2DCFDA [5 (and-6)-Carboxy-2′, 7′-dichloro-dihydroflourescein diacetate)] as the probe (http://probes.invitrogen.com). Carboxy H2DCFDA was used because it carries an additional negative charge that improves its retention compared to non-carboxylated forms (http://probes.invitrogen.com/media/pis/g002.pdf). Intracellular oxidation of Caroxy H2DCFDA to DCF (Figure 2.10) was monitored according to the increase in fluorescence as measured by a plate reader and using confocal fluorescence microscopy.

Carboxy-H2DCFDA

Oxidised product DCF

Oxidative stress

Figure 2.10. Conversion of Caroxy H2DCFDA to DCF

In brief, the assay was performed in black 96 well microplates (Nunc, Denmark). The J774A.1 cells were seeded in 100 l of cell suspension in each well at a density of 4 x 105 cells/ml and at 8 x 105 cells/ml for PLHC-1 cells. After 24 h of cell attachment, plates were washed with 100 µl/well PBS and the cells were treated with increasing concentrations of each generation of dendrimer prepared in 5% FCS containing media. Hydrogen peroxide (400 M) was used as positive control to validate the protocol. All incubations were performed at 37°C in a 5% CO2 humidified incubator. Six replicate wells were used for each control and test concentrations per 96 well microplate. After the specified incubation time period (1, 2, 4 and 6 h) the plates were washed with 100 µl/well PBS and then 100 µl/well of 10 µM Carboxy H2DCFDA was added to each well. The plates were incubated at 37°C for a period of 40 minutes. The fluorescence was quantified using a plate reader, which provides an average of the statistically variable response of individual cells (Elbekai and El-Kadi, 2005). Fluorescence was measured using an excitation of 485nm and emission of 530nm, in a TECAN GENios (Grodig, Austria) microplate reader. For visualisation of the intracellular fluorescence, carboxy H2DCFDA was excited at 488 nm and fluorescence emission at 520 nm (with a 505 nm long pass filter) was recorded using a confocal laser scanning microscope (LSM 510 META, Zeiss, Germany).  Fluorescence and phase contrast images were recorded from a minimum of 3 areas per sample.

2.2.2.10 Cytokines assay

An enzyme linked immunosorbant assay (ELISA) was performed to quantify the proinflammatory mediators (IL-6, TNF- and MIP-2) after the exposure of the J774A.1 cells to PAMAM dendrimers. The basic principle of ELISA is shown in figure 2.11 . LPS (lipo-polysaccharide) was used as positive control to stimulate the TNF- and MIP-2 and validate the ELISA protocol. The principle of the ELISA is based on the sandwich technique, in which the capture antibody (primary antibody) at concentrations of 1g/ml (TNF-), 2 g/ml (IL-6) and 0.5 g/ml (MIP-2) in PBS (pH -7.4), was coated in the 96 well plate (Nunc-immuno plate, Denmark).

Figure 2.11. Principle of ELISA (Sandwich technique)

(http://www.synchronium.net/wpcontent/uploads/2009/09/sandwich_elisa.jpg&imgrefurl)

The plates were incubated overnight at room temperature. The wells were aspirated to remove the liquid and the plates were washed four times with PBS-T (phosphate buffer saline with 0.05% of Tween 20) and then blocked with 1% BSA solution at room temperature for 1 hour. The plates were again washed with PBS-T four times and 100 l of different dilutions of supernatant were added to the respective wells and standards of IL-6, TNF- and MIP-2 at a concentration from 10 to 800 pg/ml in duplicate were added to the first two columns of the 96 well plates and incubated for 2h at room temperature. The plates were aspirated and washed four times, whereupon 100l of the detection antibody against the respective marker (secondary antibody) were added to the 96 well ELISA plate at a concentration of 0.25g/ml (for TNF- and MIP-2), or 0.5 g/ml (IL-6) and the plates were incubated at room temperature for 2h. The plates were aspirated and washed four times, 100 l of avidine-HRP (1:2000 dilutions in blocking buffer) were added to each well and the plates were incubated for 30 minutes at room temperature. The plates were washed four times with washing buffer and 100 l of substrate solution (2,2'-Azino-Bis(3-Ethylbenzthiazolin-6-Sulfonic acid)) were added to each well and the plates were incubated at room temperature to develop the colour. The colour development time was optimised to be 15 minutes for each assay using the standards and the absorbance was measured at 405 nm in a VICTOR3VTM 1420 Multilabel Counter plate reader (Perkin Elmer, USA).

2.2.2.11 Oxidative DNA damage

8-hydroxy-2'-deoxyguanosine (8-OHdG) is formed when DNA is oxidatively modified by ROS, as shown schematically in Figure 2.12. Oxidative stress has been demonstrated to play a potential role in the initiation, promotion, and progression of malignancy. Lesions such as 8-OHdG are coupled with their potential mutagenicity in mammalian cells, and this has led to their proposed potential as intermediate markers of a disease endpoint for example, cancer.

8-hydroxyguanosine

Figure 2.12. Schematic diagram of the conversion of Deoxyguanosine to 8- hydroxyguanosine.

2.2.2.11.1 DNA Extraction from PLHC-1 cells

DNA was extracted using the DNA extractor WB kit (Wako pure chemicals Industries, LTD, Osaka, Japan). In brief, the cells were plated in a T-25 culture flask (Nunc, Denmark), at a seeding density of 1x106 and kept for 24h to allow for attachment. They were then exposed to different concentration of PAMAM dendrimer solutions for the different time points (6, 12, 24, 48 and 72h). The exposure was terminated after the appropriate exposure time by removing the medium and rinsing with PBS. The cells were then trypsinized and centrifuged to remove the supernatant, 0.5 ml of lysis solution was added to the pellet and the suspension was mixed gently by inversion of the microfuge tube. The cell suspension was then centrifuged at 10,000x g for 20 seconds at 4 oC. One millilitre of lysis solution was then added to the pellet and the suspension was again mixed gently by inversion of the microfuge tube, and subsequently centrifuged at 10,000x g for 30 second at 4 oC. The lysis step was repeated one more time.

The resultant pellet was suspended in 200 l of enzyme reaction solution and 10 l of protease solution was added and the suspension was mixed gently by inversion. The reaction mixture was incubated at 37 oC for 1 hour and the solution was mixed several times by inversion. After the incubation time, 0.3 ml of sodium iodide followed by 0.5 ml of isopropyl alcohol was added to the reaction mixture and the solution was mixed by inversion of the microfuge tube until a whitish material appears. It was then centrifuged at 10,000 g for 10 minutes at room temperature. The pellet was rinsed with washing solution A and then washing solution B. The pellet was reconstituted in MQ water and maintained at 4 oC. The purity of the extracted DNA was determined by UV-visible spectroscopy at 260 and 280 nm. The absorbance value of the ratio of 260/280 nm was obtained ~ 1.8, which indicates that the extracted DNA is pure.

After DNA extraction, the DNA was digested for the determination of 8-OHdG by the ELISA method. The DNA was converted to single strand by incubating the sample at 95 oC for 5 minutes and then rapidly chilling on ice. The DNA sample was then digested to nucleotides by incubating the denatured DNA with 5 units of nuclease P1 for 2 hour at 37 oC in 20 mM Sodium Acetate, pH 5.2. Subsequently it was treated with 5 units of alkaline phosphatase for 1 hour at 37 oC in 100 mM Tris buffer, pH 7.5. The reaction mixture was centrifuged for 5 minutes at 6000 g and the supernatant was used for the 8-OHdG assay.

2.2.2.11.2 Measurement of 8-OHdG by ELISA

The 8-OHdG ELISA kit is a competitive in vitro enzyme linked immuno-sorbent assay for quantitative measurement of the oxidative DNA adduct 8hydroxy2' deoxyguanosine (8-OHdG). All reagents and samples were equilibrated to room temperature before use (20-25oC). The ELISA was carried out according to the manufacturer's instructions. In brief, the primary antibody was reconstituted with the primary antibody solution and allowed to dissolve completely.

Fifty microlitres of sample (extracted DNA) or standard was added per well, and then 50μl of reconstituted primary antibody was added per well. The plate was shaken from side to side and the solution mixed fully. The container was covered with adhesive strip, making sure it was sealed tightly, and incubated at 4 oC overnight. The contents of the plate were removed. Two hundred and fifty microlitres of washing solution were pipetted into each well. After washing thoroughly by shaking the plate from side to side, the washing solution was removed. The plate was inverted and blotted using a clean paper towel to remove any remaining washing buffer. The washing process was repeated twice more.

The secondary antibody was reconstituted with the secondary antibody solution, dissolving completely. One hundred microlitres of constituted secondary antibody was added per well. The plate was shaken from side to side to mix fully. The plate was covered with an adhesive strip and incubated at room temperature for 1 hour. At the end of the incubation period, the plates were washed twice with washing buffer. The chromatic solution (enzyme substrate solution) was reconstituted with 100 times the volume of the diluting solution. One hundred microlitres of the reconstituted enzyme substrate was added per well. The plate was shaken from side to side to mix fully and incubated at room temperature for 15 minutes in the dark.

One hundred microlitres of the reaction terminating solution was added per well. The plate was shaken from side to side to mix fully. After terminating the reaction, the absorbance at 450 nm was measured. A standard curve was used to determine the amount of 8-OHdG present in test samples.

2.2.2.12 Alkaline Comet assay

The alkaline comet assay, also known as the 'single cell gel electrophoresis' (SCGE), is a sensitive and rapid technique for quantifying and analysing DNA damage in individual cells, such as a single and double strand breaks and alkali-labile sites in the living cells (Collins et al., 2004). The resulting image that is obtained resembles a comet with a distinct head and tail. The head is composed of intact DNA while the tail consists of damaged (single stranded or double stranded breaks) or fragments of DNA. For the analysis by the comet assay, individual cells are embedded in a thin agarose gel on a microscope slide. All cellular proteins are then removed from the cells by lysing. The genomic DNA is allowed to unwind under alkaline/neutral conditions. Following the unwinding, the DNA undergoes electrophoresis, allowing the broken DNA fragments or damaged DNA to migrate away from the nucleus. After staining with DNA specific fluorescent dye, the gel is read for the amount of fluorescent in head and tail and the length of tail. The extent of DNA liberated from the head of the comet is directly proportional to the amount of DNA damage.

The olive tail moment (OTM) is one of the most important parameters and is calculated as the product of two factors: the percentage of DNA in the tail (tail percentage DNA) and the distance between the intensity centroid of the head (head mean) and the tail (tail mean) along the x-axis of the comet. It is calculated by the formula-

Olive Tail Moment (OTM) = (Tail.mean - Head.mean) X %Tail DNA/100.

Figure 2.13. Comet measurement parameter, Head length is the distance from blue line to green line. Tail length is the distance from green line to pink line. Head intensity is the number of pixels under the symmetric curve. Tail intensity is the number of pixel under the skewed curve to the right drawn in orange.

The genotoxicity of NIPAM nanoparticles was assessed using the micro-comet assay technique in three cell lines (HaCaT, SW480 and PLHC-1 cells). For a typical experiment, 100 l of 1x105 cells/ml for 24h; 8Ã-104 cells/ml for 48h; 6Ã-104 cells/ml for 72h exposure of nanoparticles were plated in 96 well microplate and incubated at 37°C in 5% CO2 for 24 hours to ensure cell attachment. The PLHC-1 cells were incubated at 30 °C during whole the experimental time period. The cell monolayers were then washed with PBS and exposed to varying particle concentrations (12.5 mg/l, 25 mg/l, 100 mg/l, 200 mg/l, 400 mg/l, and 800 mg/l) for different time intervals (24, 48 and 72h). For PAMAM dendrimers, cells were exposed to different concentrations of G-4, G-5 and G-6 for 6, 12, 24, 48 and 72h. After the appropriate exposure time, cells were washed once with PBS, trypsinized and suspended in low melting point agarose and cast onto a gel bond film fixed with chamber slides. After the agarose solidified, it was suspended in freshly prepared and pre-cooled cell lysis buffer overnight. The following day, electrophoresis was conducted in alkaline electrophoresis buffer (pH 12.7) for 15 mins (conditions: 300 mA, 1.5 V/cm at 4°C). After completion of the electrophoresis run time, the Gelbondâ„¢ film was treated with neutralisation buffer (pH 7.5) for 30 minutes to neutralise the DNA embedded gels and then dehydrated in absolute ethanol for 2 h. Gels were stored in the dark overnight at 4°C, allowed to dry completely, and were then stained with SYBR-Green nucleic acid stain. Image analysis was performed using Komet 5.5 software (ANDORâ„¢, UK) and a Nikon Eclipse E600 microscope attached to a CCD camera (Figure 2.13). Values of OTM and percentage of tail DNA were automatically calculated by the software. Ethyl Nitrosourea (ENU) was used as a positive control to validate the experimental protocol.

2.2.2.13 Apoptosis assay

Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. Inappropriately regulated apoptosis is implicated in disease states, such as Alzheimer's disease and cancer. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes, including compaction and fragmentation of the nuclear chromatin, shrinkage of the cytoplasm and loss of membrane asymmetry (Barnden et al., 1998; Darzynkiewicz et al., 1997). Furthermore, during apoptosis the cytoplasmic membrane becomes slightly permeant. Certain dyes, such as the green fluorescent YO-PRO®-1 dye can enter apoptotic cells, whereas other dyes, such as the red fluorescent dye, propidium iodide (PI), cannot. Thus, use of YO-PRO®-1 dye and PI together provide a sensitive indicator for apoptosis (Idziorek et al., 1995; Estaquier et al., 1996). The Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO®-1 and PI for flow cytometry provides a rapid and convenient assay for apoptosis. The kit contains ready-to use solutions of both YO-PRO®-1 and PI dyes. After staining a cell population with YOPRO®-1 dye and PI, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished by a flow cytometer that uses the 488 nm line of an argon-ion laser for excitation (Figure 2.14).

Figure 2.14. CyFlow® space (http://www.google.ie/images)

The PLHC-1 cells were plated in a 6 well plate at a seeding density of 1 x 106 cells/ml well. The plates were incubated at 30 oC for 24 hour to ensure proper attachment. The cell monolayer were washed with PBS and then exposed with a range of concentration of PAMAM dendrimers (G-4, G-5 and G-6) for different time points (6, 12, 24, 48 and 72h). After the appropriate exposure time, cells were washed once with PBS, trypsinized, centrifuged, the supernatant removed and then the cell pellets were suspended in 1 ml PBS. One microlitre of YO-PRO®-1 dye and 1l PI were added to the cell suspension and it was incubated on ice for 30 minutes. After the incubation time, the fluorescence of the cell suspension was measured in flowcytometer (CyFlow® space). The experimental protocol was validated by using camptothecin as positive control.

2.2.2.14 Statistics

All experiments were conducted in at least triplicate (three independent experiments). Toxicity was expressed as mean percentage inhibition for the Microtox® (bioluminescence), D. magna (immobilisation) and percentage mortality was measured for the T. platyurus assay. Fluorescence (AB assay) as fluorescent units (FUs) was quantified using a microplate reader (TECAN GENios, Grödig, Austria). Raw data from cell cytotoxicity assays were collated and analyzed using Microsoft Excel® (Microsoft Corporation, Redmond, WA). Cytotoxicity and the intracellular ROS (Reactive oxygen species) were expressed as mean percentage inhibition relative to the unexposed control ± standard deviation (SD). MIP-2, IL-6 and TNF-α data were calculated from their respective standards and were expressed in mean (pg/ml) ± standard deviation (SD). The genotoxicity assay was performed twice in duplicate, and was expressed in terms of percentage tail DNA and OTM as the mean percentage ± standard deviation (SD). Statistical analyses were carried out using one-way analyses of variance (ANOVA) followed by Dunnett's multiple comparison tests. Statistical significance was accepted at P ≤ 0.05 for all tests. Toxicity data was fitted to a sigmoidal curve and a four parameter logistic model used to calculate EC/LC50 values. This analysis was preformed using Xlfit3â„¢ a curve fitting add-in for Microsoft® Excel (ID Business Solutions, UK).

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