Chapter Materials and Experimental Methods Below are the materials and methodology used in the course of the research project. Included are the methods for polymer synthesis and characterization, fabrication and characterization of micelles (i.e. core-shell nanoparticles), drug loading, fabrication and characterization of micelles/DNA complexes, and in vitro as well as in vivo gene transfection. 3.1 Polymer synthesis 3.1.1 Materials Cholesteryl chloroformate (98%), Aldrich, USA N-methyldiethanolamine (99%), Aldrich, USA Adipoyl chloride (98%), Aldrich, USA Sebacoyl chloride (97%) Aldrich, USA 2-Bromoethylamine hydrobromide (>99%), Sigma, USA Triethylamine (≥99%), Sigma, USA Monomethyoxy poly(ethylene glycol) (mPEG) ( or polyethylene glycol monomethyl ether) MW 5000, 2000, 1100 or 550 Da), Sigma, USA. Tetrahydrofuran (THF), ACS grade, Tedia or Merck, USA Diethyl ether, ACS grade, Tedia or Merck, USA 39 Toluene, ACS grade, Tedia or Merck, USA Chloroform, ACS grade, Tedia or Merck, USA Anhydrous ethanol, Merck, USA Andydrous acetone, Merck, USA Anhydrous sodium carbonate, Sigma, USA Hexane, ACS, Merck, USA Methanol, ACS, Merck, USA Magnesium sulfate, Merck, USA 37% Hydrochloride solution, Sigma, USA Sodium chloride, Sigma, USA Sodium, Merck, USA Molecular sieve Sigma, USA Benzophenone, Sigma, USA p-Toluenesulphonyl chloride, Lancaster, England Dialysis membrane, Spectra/Pro, MWCO3500, 8000, USA 25 Thin layer chromatograph plastic sheet, Silica gel 60F254, 20*20cm, Merck, USA N-Methyldiethanolamine, adipoly chloride, and sebacoyl chloride were purified by distillation under vacuum. Triethylamine was treated with toluene sulphonyl chloride to remove primary and secondary amine. It was then distilled and freshly dried with sodium prior to synthesis. THF was freshly dried with sodium and distilled before use. Benzophenone was used as the indicator that the moisture has been removed completely. Toluene was dried by sodium before use. Chloroform was dried by molecular sieve prior to use. The rest chemicals were used as received. 40 3.1.2 Synthesis of N-(2-bromoethyl) carbarmoyl cholesterol (Be-chol) 50 mL of chloroform dried in molecular sieves were put into a 100-mL round-bottom flask in a dry ice/acetone bath (temperature: lower than -30◦C). 4.34 g of cholesteryl chloroformate (0.0097 mol) and 2.18 g of 2-bromoethylamine hydrobromide (0.0106 mol) were then added with stirring. Next, mL of freshly dried triethylamine were added to the flask. Then the dry ice/actone bath was moved after half an hour for the reaction to proceed at room temperature for 12 hours. The organic solution was washed times with 20 mL of N HCl solution saturated with NaCl, and once with 30 mL of NaCl-saturated aqueous solution to remove residual triethylamine. The organic phase was collected and dried with g of anhydrous magnesium sulfate. The solution was then filtered and distilled. The crude product was recrystallized with anhydrous ethanol once and with anhydrous acetone twice. The final product was dried in a vacuum oven for 24 hours. The yield was ~ 78%. The thin layer chromatography (TLC) test showed its flow ratio (Rf) was 0.68 in the solvent mixture of toluene, hexane and methanol (8:8:1 in volume). The synthetic route is shown in Scheme 1. 3.1.3 Synthesis of poly(N-methyldietheneamine sebacate) (PMDS) and poly(Nmethyldietheneamine adipate) (PMDA) 5.958 g of N-methyldiethanolamine (0.05mol) and 50.5 g of triethylamine (0.5mol) were added to 150-mL of round-bottom flask in a dry ice/acetone bath (below -30°C). 40 mL of THF (dried with sodium) containing 11.945 g of sebacoyl chloride (0.05 mol) were added drop wise to the flask with stirring. The flask was removed hour later, and the reaction was allowed to proceed at room temperature for more days. The solution 41 was filtered and harvested. The solid was washed three times with 300 mL of THF and the solution was also collected by filtration. The solvent was then removed using the rotavapor. The crude product was semi-solid, which was put in a vacuum oven overnight to further remove triethylamine. Thereafter, the crude product was dissolved in 150 mL of toluene and washed three times with 45 mL of NaCl-saturated aqueous solution, pH of which was adjusted to ~ with sodium carbonate. The toluene solution was then dried with anhydrous NaCO3. Toluene was removed using the rotavapor and the product was dried in the vacuum oven for two days. The yield was ~ 40%. Poly(N-methyl dietheneamine adipate) was synthesized by a similar protocol as described above. The synthetic route is shown in Scheme 2. 3.1.4 Synthesis of poly{(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate} (P(MDS-co-CES)) and poly{(N-methyldietheneamine adipate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] adipate} (P(MDA-co-CEA)) 2.85 g of PMDS (0.01M repeat unit) and 5.5 g of N-(2-bromoethyl) carbarmoyl cholesterol (Be-chol) (0.01 mol) were dissolved in 50 mL of dry toluene and refluxed for days under argon. The solution was distilled using the rotavapor to remove toluene; 100 mL of diethyl ether were then added to precipitate the product. To completely remove unreacted N-(2-bromoethyl) carbarmoyl cholesterol, the product was washed with diethyl ether more times. The yield was 30% to 70%. Poly{(N-methyldietheneamine adipate)-co-[(cholesteryl oxocarbonylamido ethyl) 42 methyl bis(ethylene) ammonium bromide] adipate} (P(MDA-co-CEA)) was synthesized by a similar protocol as presented above. The synthetic route is shown in Scheme 2. 3.1.5 PEGylation of PMDS 5.958 g of N-methyldiethanolamine (0.05mol), 0.00125 mol of mPEG (6.25 g, 2.5 g, 1.375 g, 0.8125 g for Mn of 5000, 2000, 1100 and 650 Dalton respectively) and 50.5 g of triethylamine (0.5mol) were added to 150-mL round-bottom flask in a dry ice/acetone bath (below -30°C). 40 mL of THF (dried with sodium) containing 11.945 g of sebacoyl chloride (0.05mol) were added drop wise to the flask with stirring. The flask was removed hour later, and the reaction was allowed to proceed at room temperature for more days. The solution was filtered and harvested. The solid was washed three times with 300 mL of THF and the solution was also collected by filtration. The solvent was then removed using the rotavapor. The crude product was semi-solid, which was put in a vacuum oven overnight to further remove triethylamine. Thereafter, the crude product was washed by using ether to remove the oligomers and triethylamine residues and dried under vacuum overnight. PEG550-PMDS, PEG1100-PMDS and PEG2000-PMDS were dissolved in acetone and dialyzed against acetone using a dialysis membrane with a molecular weight cut-off of 3.5 kDa for two days to further remove the unreacted PEG and other impurity. PEG5000-PMDS were dialyzed using a dialysis membrane with a molecular weight cut-off of kDa. The yield of PEGylated PMDS is ~ 70%. The PEGylated PMDA was synthesized by a similar protocol presented above. The schematic route is described in Scheme 3. 43 3.1.6 Synthesis of PEGylated P(MDS-co-CES) PEGylated PMDS was first characterized by using 1H-NMR to determine the ratio of PEG block to PMDS block, and the amount of Be-chol was added according to the amount of PMDS units in a molar ratio of 1:1. For example, the content of PMDS calculated by 1H-NMR was 80% in weight. Thus, 2.5 g of PEGylated PMDS contained 2.0 g of PMDS, i.e. 0.007mol of repeated PMDS units (2.0/285=0.007). Therefore, the amount of Be-chol added was 3.74g (0.007×532.9=3.74). PEGylated PMDS and Be-chol were dissolved in 100 mL of dry toluene and refluxed for 24 hr under argon. Toluene was removed by distillation using the rotavapor. The crude product was washed with diethyl ether four times to remove unreacted N-(2-bromoethyl) carbarmoyl cholesterol and dried overnight in a vacuum oven. The yield was ~ 50%. The schematic route is described in Scheme 3. H3C CH H3C H3C CH H triethylamine O BrCH 2CH 2NH + H H Cl O cholesteryl Chloroformate bromoethylamine H3C CH H3C H3C CH H O H Br NH H O N-(2-bromoethyl)carbarmoyl cholesterol(Be-chol) Scheme Synthesis of N-(2-bromoethyl)carbarmoyl cholesterol (Be-chol). 44 O CH O Cl-C (CH 2)n C -Cl sebacoyl chloride(n=8) adipoyl chloride (n=4) O HO (CH 2)2 + triethylamine (CH 2)2 N OH N-methyldiethanolamine CH O C (CH 2)n C O (CH 2)2 N Be-chol (CH 2)2 O CH3 H3C m poly(N-methyldietheneamine sebacate)(PMDS)(n=8) poly(N-methyldietheneamine adipate)(PMDA)(n=4) CH3 H3C H H3C H H O O NH O O C (CH2)n C CH O (CH2)2 N (CH2)2 O O O C (CH2)n C O (CH2)2 Br + N (CH2)2 q O CH3 p P(MDS-co-CES)(n=8) P(MDA-co-CEA)(n=4) Scheme Synthesis of P(MDS-co-CES) and P(MDA-co-CEA). O CH3 O Cl-C (CH 2)n C -Cl + HO (CH 2)2 N (CH 2)2 OH + CH3-(OCH2CH2)S-OH sebacoyl chloride(n=8) N-methyldiethanolamine mPEG adipoyl chloride (n=4) O CH3-(OCH2CH2)S-O triethylamine CH3 O C (CH 2)n C O (CH 2)2 N (CH 2)2 (CH2CH2O)S-CH3 O CH3 H3C m poly(N-methyldietheneamine sebacate)(PMDS)(n=8) poly(N-methyldietheneamine adipate)(PMDA)(n=4) CH3 H3 C H H3 C H Be-chol O O O C (CH2)n C CH O (CH2)2 N (CH2)2 O H O NH O O C (CH2)n C O (CH2)2 q Br + N (CH2)2 CH3 O p Pegylated P(MDS- co-CES)(n=8) Pegylated P(MDA- co-CEA)(n=4) Scheme Pegylation of P(MDS-co-CES) and P(MDA-co-CEA). 45 3.2 Characterization of polymers 3.2.1 Materials Pyrene (≥99.0%), Fluka, USA Anhydrous acetone, AR grade, Merck, USA D-chloroform, AR grade, Sigma, USA Chloroform, HPLC grade, Merck, USA Tetrahydrofuran, HPLC grade, Merck, USA Phosphate -buffered saline (PBS) 10X, Sigma, USA, diluted to time 3.2.2 1H-NMR analysis The 1H-NMR spectra of the polymers were recorded on a Bruker AVANCE 400 spectrometer (400MHz). Chemical shifts were expressed in parts per million (δ) using residual protons in the indicated solvent as the internal standard. 3.2.3 FTIR analysis The FTIR spectra of the polymers were analyzed using a Fourier transform infrared spectrometer (Perkin Elmer Spectrum 2000, USA). The polymer was dissolved in chloroform, and the solution was then dropped onto the sodium chloride crystal cell. The solvent was allowed to evaporate completely prior to the measurements. 3.2.4 Gel permeation chromatography (GPC) analysis 46 The molecular weights of PMDS, PMDA, P(MDS-co-CES) and P(MDA-co-CEA) were determined by GPC (Waters 2690, MA, USA) with a differential refractometer detector (Waters 410, MA, USA). 10 mg of polymer was dissolved in mL of THF and the solution was then filtered. The mobile phase was THF with a flow rate of mL/min. Weight and number average molecular weights were calculated from a calibration curve using a series of polystyrene standards (Polymer Laboratories Inc., MA USA, with molecular weight ranging from 1300 to 30,000). 3.2.5 TGA analysis Thermalgravimetric analysis of the polymers was carried out using a thermogravimetric analyzer (TGA, Perkin Elmer TGA 7, USA) under air, and the temperature rising rate was 20ºC/min. The temperature scanning range was between 30ºC and 700ºC. 3.2.6 DSC analysis Glass transition temperature (Tg) of the polymers was measured using a TA 2920 modulated differential scanning calorimeter (DSC) (Perkin-Elmer, CT, USA). The temperature of DSC had been calibrated with an indium standard. The glass transition temperature (Tg) was determined by first cooling the sample from 30 to –10°C and then heating to 120°C at a heating rate of 10°C/min in a nitrogen atmosphere. 3.2.7 Elemental analysis 47 The nitrogen content of the polymers was determined by elemental analysis using Perkin-Elmer Instruments Analyzer 2400. 3.2.8 Polymer degradation study The degradation of the polymers was studied by recording their weight loses in PBS (pH 7.4) at predetermined time intervals. A fixed mass of polymer was put in mL of PBS and the mixture was incubated at 37ºC. The solution was changed with fresh PBS every 24 hr. The samples were taken out at Day 3, 7, 14, 28 and 56, and freeze dried for two days before being weighed. 3.2.9 Determination of critical micelle concentration (CMC) CMC of polymers was estimated by fluorescence spectroscopy using pyrene as a probe. Aliquots of pyrene solution (10 µg/mL in acetone, 400µL) were added to 5-mL volumetric flasks, and the acetone was allowed to evaporate. mL of aqueous polymer solutions of 0.1–50 mg/L were then added to the volumetric flasks containing the pyrene residue, so that the solutions all contained excess pyrene at a concentration of 0.1 µg/mL. The solutions were allowed to equilibrate for 20 hours at room temperature followed by hours at 60ºC before fluorescence spectra were obtained using a LS50B luminescence spectrometer (Perkin Elmer, U.S.A.). The excitation spectra (300–360 nm) were recorded with an emission wavelength of 395 nm, and the emission spectra (360-410 nm) were recorded with an excitation wavelength of 339 nm. The excitation and emission bandwidths were set at 4.5 nm. The ratios of the peak intensities at 338 nm and 333 nm (I338/I333) from the excitation spectra and I3 (the third peak at 385nm)/I1 (the first peak at 48 spectrometer. For the pyrene, paclitaxel and verapamil-loaded micelles, 100 µL of pyrene-loaded micelle solution was mixed with mL of DMF and measured directly by the UV-VIS spectrometer. The mixture of DMF and buffer solution was used as reference. The detection wavelength was set at 318 nm for indomethacin, 273 nm for pyrene, 266 nm for paclitaxel, and 277nm for verapamil. The standard curves were obtained by preparing standard indomethacin pyrene, paclitaxel and verapamil solutions with different concentrations in DMF. The loading level of cyclosporin A was measured by HPLC. Briefly, the cyclosporin A loaded nanoparticles solution was firstly freeze-dried and then dissolved in 1ml ethanol and then filtered by using 0.2μm of filter paper and analyzed for CyA levels using high-performance liquid chromatography (HPLC). The HPLC system consisted of a Waters 2690 separation module and a Waters 996 PDA detector (Waters Corporation, USA). A Waters SymmetryShieldTM C8 4.6×15.0 cm column fitted with a C8 pre-column was used. The mobile phase isopropanol with column and sample temperatures set at 50°C and 15°C, respectively. The detection wavelength was set at 210 nm. The retention time was 3.2±0.1 min. A calibration curve was constructed to determine CyA concentration in the range from to 20 ppm and the r2 value was at least 0.999. The encapsulation efficiency was calculated as the ratio of the actual drug mass encapsulated to the initial drug mass added. The loading level was calculated as the ratio of loaded drug mass to the total mass of polymer and loaded drug. 3.5 Binding of DNA with blank and drug-loaded polymeric micelles 3.5.1 Materials Agarose, biological grade, Bio-Rad, USA 52 Ethidium bromide, 10mg/ml, Sigma, USA DNA loading buffer, times, Sigma, USA Tris-Acetic acid-EDTA Buffer Solution (TAE), 10 times, Sigma, USA, diluted to times before using Sodium chloride, ACS grade, GCE, USA DNA encoding the 6.4 kb firefly luciferase (pCMV-luciferase VR1255_C) driven by the cytomegalovirus (CMV) promoter/enhancer (luciferase-plasmid) was kindly provided by Prof. K. W. Leong’s laboratory at Johns Hopkins Singapore, and amplified by using Qiagen Endofree® Plasmid Giga Kit. 3.5.2 Agarose gel electrophoresis measurements The DNA binding ability of the blank and drug-loaded polymeric micelles was analyzed by agarose gel electrophoresis. The blank and drug-loaded micelles/DNA complexes containing 0.28 µg of luciferase-plasmid were prepared at various N/P ratios. The N/P ratio means the ratio of amine groups in the cationic polymer, which represent the positive charges, and phosphate groups in the plasmid DNA, which represent the negative charges. The complexes solutions at various N/P ratios were diluted to an identical volume (i.e. µL) by using the same buffer employed for preparation of the micelles. µL of times DNA loading buffer was added to the complexes solutions. The mixtures were allowed to stay at room temperature for 45 minutes. Thereafter, the complexes were loaded into individual wells of 1.0 % agarose/1×TAE gel containing 0.5 µg/mL ethidium bromide and electrophoresised at 100 V for 90 minutes. The naked DNA diluted with the same buffer without adding the micelles and the micelles without adding 53 DNA were used as the controls. The tracks of the DNA and complexes were observed under UV transilluminator (vilber lourmat, France) and the photos were taken. 3.5.3 Competition binding assays The DNA binding ability of polycations can also be analyzed by dye-exclusion assays [Wolfert M. et al., 1996]. To stain the luciferase-plasmid with ethidum bromide (EtBr), Triplicate mixtures of 50μl of luciferase DNA (40 μg/ml) and 50 μl of ethidum bromide (0.8μg/ml) were added into 96well plate and allowed to incubate in room temperature for 30mins first. Fluorescence (λex=355nm, λem=590nm) of DNA/ethidum bromide complexes solution was measured by fluorescence microplate reader (spectra MAX GEMXS, molecular devices, USA) and set as 100% of fluorescent intensity against 100 μl of naked DNA solution without adding ethidium bromide (triplicate). Aliquots of cationic polymeric micelles (0.5-1 μl) or drug loaded polymeric micelles were added stepwise into the DNA/ethidium bromide solution and the naked DNA solution without adding ethidium bromide (as background at each step), with gentle mixing, and fluorescence levels were allowed to stabilize for 5mins before measurement at each step. The real fluorescence change at each step was calculated by subtracting the related background fluorescence at each step. The procedure was continued until no obvious decrease of fluorescent intensity was observed. The percentage decrease of fluorescent intensity was allowed to be calculated as function of N/P ratios. The binding competition experiment described at the previous section was continued by adding 1μl, of 5M sodium chloride aqueous solution stepwise to study the influence of ionic strength on DNA binding of with polymeric micelles. Similarly, the sodium 54 chloride solution was added gently and mixed with the substrate. The fluorescence levels of the complexes were allowed to stabilize for 5mins at each step before measurement. Stop adding sodium chloride when there was no obvious increase of the fluorescent intensity. 3.6 Stability of drug-loaded micelles/DNA complexes 3.6.1 Preparation of the complexes for the particle size and zeta potential analysis The pyrene-loaded micelles were fabricated by dialysis against sodium acetate/acetic acid buffer (0.02M, pH 4.6) using a membrane with a molecular weight cut-off of kDa and filtered by 0.45 µm filter. The solution was added gradually into mL of the sodium acetate/acetic acid buffer (0.02M, pH 4.6) containing 40 µg of DNA. The mixture was then vortexed for minutes before analysis of particle size and zeta potential. 3.6.2 Structural integrity of drug-loaded micelles after DNA binding It is known that the I3/I1 ratio from the emission spectra (λex=339 nm) of pyrene and the I338/I333 ratio from the excitation spectra (λem=395nm) of pyrene change with changing the polarity of the microenvironment of pyrene [Jones M-C., 1999]. When pyrene enters a more hydrophobic environment, the ratios increase. To study the structural integrity of the pyrene-loaded micelles after DNA binding, the ratios were measured before and after DNA binding. Freshly prepared pyrene-loaded micelles as described in Section 3.7.1 were diluted 300 times with the sodium acetate buffer (0.02M, pH 4.6). A certain amount of the plasmid was added gradually into the micelle solution. 55 The mixed solution was vortexed for 15 minutes prior to fluorescence analysis. For the details of fluorescence analysis, please refer to Section 3.2.9. 3.7 In vitro drug release from the drug-loaded polymeric micelles and micelles/DNA complexes In vitro release of indomethacin from the indomethacin-loaded micelles and the micelles/DNA complexes were performed to investigate the effect of DNA binding. The indomethacin-loaded micelles were prepared as described in Section 3.4.2. The loading level and loading efficiency were measured as described in Section 3.4.3. The indomethacin-loaded micelles prepared in the sodium acetate/acetic acid buffer (0.02M, pH 4.6) were mixed with the buffer containing luciferase-plasmid at the N/P ratio of 15. The indomethacin-loaded micelles or the micelles/DNA complexes were then put in a dialysis membrane with a molecular weight cut-off of kDa. The dialysis membrane was placed in 50 mL of PBS (pH 7.4) at 37 °C. At fixed time intervals, the external phase was sampled and analyzed for indomethacin level using the UV-VIS spectrometer at 318 nm. 3.8 Culture of cells 3.8.1 Cell lines and materials HepG2, Hek293, Hela, 4T1, KB-31-MA, L929, Human dermal fibroblast, purchased from ATCC, USA Dulbecco’s modified Eagle’s medium (DMEM), Sigma, USA Roswell Park Memorial Institute 1640 medium (RPMI 1640), Sigma, USA L-glutamine, 200mM, IRVINE Scientific, USA 56 Penicillin-streptomycin solution, 10,000 units of penicillin and 10mg streptomycin in 0.9% NaCL, Sigma, USA Fetal bovine serum, Sigma, USA Trypsin-EDTA 10 times, Sigma, diluted to time 3.8.2 Maintenance of cells HepG2, Hek293, Hela, L929, KB-31-MA, human dermal fibroblast cell lines were maintained in Dulbbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, mM of L-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37°C under an atmosphere with 5% CO2 using 75ml plastic flask. 4T1 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640) supplemented with 10% FBS, mM of L-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37°C under an atmosphere with 5% CO2. To subculture the confluent cells, the medium was removed first and washed with 5ml PBS buffer and then detached by 1ml trypsin. 1/5 of the cells were passed to next flask. 3.9 Cytotoxicity of the micelles and the micelles/DNA complexes 3.9.1 Material 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Sigma, USA, dissolved in PBS at concentration of 0.5mg/ml and sterilized using 0.22μm filter. Dimethyl sulfoxide (DMSO), biological grade, Sigma, USA 3.9.2 Polymeric micelles 57 The micelles were prepared in de-ionized water by the same method described in Section 3.3.2. The accurate concentration of the micelles after filtration using 0.22 µm filter was determined by weighing the freeze-dried micelles. The micelle solution was diluted to different concentrations to perform the cytotoxicity test. To test the cytotoxicity of polymeric micelles or polymeric micelles/DNA complexes against different cell lines, the cells were seeded onto 96-well plates at 5000 cells per well. The plates were then returned to the incubator. In the morning of the tests, the media in the wells were replaced with 150 µL of fresh media. Each polymeric micelles solution (50 µL) was then added to each well. Sodium acetate buffer (0.02M, pH 4.6) of an equivalent volume was used as the negative control. The plates were then returned to the incubators, and maintained in 5% CO2 at 37°C for a certain period. Each sample was tested in replicates per plate. Aliquots of MTT solution (20 µL) were added into each well after the designated period. The plates were then returned to the incubator, and maintained in 5% CO2 at 37°C for hours. The growth medium in each well was removed, and 150 µL of DMSO were added to each well to dissolve the internalized purple formazan crystals. An aliquot of 100 µL was taken from each well and transferred to a new 96-well plate. The plates were then assayed at 550 nm and 690 nm using a microplate reader (PowerWave X, Bio-Tek Instruments). The absorbance readings of the formazan crystals were taken to be that at 550 nm subtracted by that at 690 nm. The results were expressed as a percentage of the absorbance of the negative control. IC50 (the concentration of the agent to inhibit the cell line growth by 50%) of the polymers were calculated based on the data obtained. 58 3.9.3 Micelles/DNA complexes The cytotoxicity of the micelles/DNA complexes at the N/P ratios of and 15 were also measured against L929 cells at the micelle concentrations of 6.0, 12.0, 24.0 and 48.0µg/mL after exposure to the cells for three days. The micelles prepared in de-ionized water as described in Section 3.4.1 were sterilized by filtration using 0.22 µm filter and then mixed with certain amount of DNA solution according to the two different N/P ratios. The complex solution was diluted to 50µl by different times according to the four different concentrations with de-ionized water and allowed to incubate at room temperature for 45 minutes. Four replicates were prepared for each sample. The complex solution was employed for the cytotoxicity test according to the protocol as described in the previous section. The PEI/DNA complexes prepared by the same method were used as the control. 3.10 In vitro gene expression 3.10.1 Materials Plasmid DNA encoding the 6.4 kb firefly luciferase (pCMV-luciferase VR1255_C) driven by the cytomegalovirus (CMV) promoter/enhancer (luciferase-plasmid), kindly provided by Prof. K. W. Leong’s laboratory at Johns Hopkins Singapore, and amplified by using Qiagen Endofree® Plasmid Giga Kit Plasmid DNA encoding the GFPmut1 variant (pEGFP-C1) with 4.7 kb driven by the SV 40 early promoter (GFP-plasmid), purchased from Clontech, USA and amplified by using Qiagen Endofree® Plasmid Giga Kit Reporter lysis buffer, 5X, Promega, USA, diluted to 1X 59 BCA Protein Assay kit, Pierce, USA Luciferase assay system, Promega, USA Paraformaldehyde, biological grade, Sigma, dissolved in PBS at concentration of 1% 3.10.2 Preparation of the micelles/DNA complexes The polymeric micelles for the in vitro study were prepared by a method similar to that described in Section 3.3.2. Briefly, 15 mg of polymer was dissolved in mL of DMF. The solution was dialyzed against 500 mL of the sodium acetate/acetic acid buffer (0.02M, pH 4.6) for 24 hours. The external phase was changed with the fresh buffer every hour for the first hours and then every hours. Thereafter, the micelle solution was harvested and the volume was measured to calculate the concentration of the micelles. Sonication was also applied to prepare cationic nanoparticles. Briefly, 15 mg of polymer was dissolved in 5-10 mL of the sodium acetate/acetic acid buffer (0.02M, pH 4.6). The solution was sonicated for 30 minutes. For preparation of the complexes, the polymeric micelle or nanoparticle solution was sterilized by filtration using 0.22µm filter and into identical amount of the plasmid solution proper amount of polymeric micelles or nanoparticles solution were added. The buffer used for the preparation of the micelles was added to dilute the complex solution to an identical volume (50 and 100 µL per well for luciferase and GFP expression, respectively). The complex solution was allowed to incubate at room temperature for 45 minutes. For in vivo gene expression experiments, the polymeric micelles and the micelle/DNA complexes were prepared using the same protocol except a higher concentration of the micelle solution was used to meet the limitation of injection volume. 60 3.10.3 In vitro luciferase expression The cells were seeded onto 24-well plates at a density of 8×104 cells per well, and cultivated in 0.5 mL of medium supplemented with 10% FBS. After 24 hours, the culture medium was replaced with fresh medium, and complexes containing 2.5 µg luciferaseencoded plasmids were added to each well. After hours of incubation, the culture media were replaced with medium containing 10% FBS. The culture media were removed after two days, and the cells on the 24-well plates were washed with 0.5 mL of phosphatebuffered saline (PBS). 0.2 mL of reporter lysis buffer was then added to each well to lyse the cells. The cell suspension was frozen in -80ºC for half hours and thawed, and then was centrifuged at 14,000 rpm for minutes. The relative light units (RLU) were measured using a luminometer (Bio-Rad, U.S.A.), and normalized to protein content using the BCA protein assay (Bio-Rad, U.S.A.). The PEI/DNA complexes at the N/P ratio of 10 were used as the positive control. Naked DNA dissolved in the same volume of the buffer was employed as the negative control. 3.10.4 In vitro GFP expression For the in vitro GFP expression, the cells were seeded onto 12-well plates at a density of 2×105 cells per well, and cultivated in mL of medium supplemented with 10% FBS. After 24 hours, the culture medium was replaced with fresh medium, and complexes containing 3.5 µg GFP-encoded plasmids were added to each well. After hours of incubation, the culture media were replaced with medium containing 10% FBS. The culture media were removed after two days. The cells on the 12-well plates were washed 61 with 1.0 mL of PBS. 0.3 mL of time trypsin was then added to each well, which was incubated at room temperature for 10–15 minutes to detach the cells. The cell suspension was centrifuged at 1,500 rpm for minutes, and re-suspended in PBS (pH 7.4). Upon separation from PBS by centrifugation, the cells were suspended in 0.3 mL of 1% paraformaldehyde for fixation prior to analyses by a cell cytometer (EPICS ELITE ESP, Coulter, U.S.A.). The GFP transfected cells in the 12-well plates were also observed under fluorescent microscope (Olympus, Japan 1X71) excited by blue light directly without any processing. 3.10.5 In vitro synergistic effect of drug and gene 3.10.5.1 In vitro synergistic effect of cyclosporin A and luciferase gene The synergistic effect of cyclosporin A and luciferase gene was performed against KB31-MA cell line, Cyclosporin A loaded polymeric micelles were prepared by dissolving 5mg of cyclosporin A and 15mg of P(MDS-co-CES) (polymer batch No. 010704) into 5mL DMF and dialyzed against 0.02M sodium acetate buffer with pH 4.6 using dialysis membrane (Spectrum, MW CUTOFF 2000) for 24 hours. 2mL of the micelles solution were freeze dried overnight and dissolved in ethanol and filtered by 0.2 µm filter paper. The ethanol solution was measured by HPLC (Waters 2690-596, MA, USA) with the mobile phase of isopropanol to determine the concentration of cyclosporin A. UV detector wavenumber was set at 210nm (see Section 3.9.3). Cyclosporin A loaded polymeric micelles/DNA complexes were prepared by the same method used for preparing polymeric micelle/DNA complexes (see Section 3.8.3). The gene transfection level of cyclosporin A loaded micelles was compared with the blank 62 micelles. In vitro gene transfection protocol was similar to that described in section 3.10.3. 3.10.5.2 In vitro synergistic effect of paclitaxel and luciferase/GFP gene The synergistic effect of paclitaxel and luciferase or GFP gene was performed agains 4T1 cell line, Paclitaxel loaded polymeric micelles were prepared by dissolving 3mg of pcalitexel and 15mg of P(MDS-co-CES) (polymer batch No. 010704) into 5mL DMF and dialyzed against 0.02M sodium acetate buffer with pH 4.6 using dialysis membrane (Spectrum, MW CUTOFF 2000) for 24 hours. The loading level of paclitaxel was measured by UV at wavenumber of 266 (see Section 3.6.3). Paclitaxel loaded polymeric micelles/DNA complexes were prepared by the same method used for preparing polymeric micelle/DNA complexes (see Section 3.8.3). The gene transfection level of paclitaxel loaded micelles was compared with the blank micelles. In vitro luciferase and GFP gene transfection protocol was similar to that described in section 3.10.3 and 3.10.4. 3.11 In vivo gene expression and synergistic effect 3.11.1 Materials Balb/C mice, provided by animal holding unit, NUS, Singapore Albino guinea pigs, provided by animal holding unit, NUS, Singapore Ketamine, biological grade, Sigma, USA Xylazine, biological grade, Sigma, USA Gel foam, biological grade, Sigma, USA 63 3.11.2 Luciferase expression in the cochlea of guinea pig 10 µL of the micelles/DNA complexes at the N/P ratio of 10 was prepared by gently mixing a micelle solution (1.35 mg/mL) with µg luciferase-plasmid in the sodium acetate/acetic acid (0.02 M, pH 4.6). The solution was allowed to stand for 30 minutes at room temperature before use. Albino guinea pigs weighing 250–300 g were used. The animals were initially anesthetized with a combination of ketamine (40 mg/kg) and xylazine (10 mg/kg). Post auricular approach was used routinely for exposure of the tympanic bony bulla. A small opening of the tympanic bulla was carefully made with forceps to provide direct visualization of the round window membrane (RWM), and a small piece of dry Gel foam was placed in the groove in direct contact with the RWM. 10.0 µL of the complexes or naked DNA (each containing µg DNA) were injected into the Gel foam, and the incision was closed in layers. Temporal bone was harvested from both sides of the head. Each bulla was opened using bone-cutting forceps to expose the cochlea. The cochlea was then scooped out using pre-cooled rongeur and immediately put into an eppendorf tube of 1.5 mL, which was then kept on ice. 200 µl of the lysis buffer (Promega, USA) was added to the tube and homogenized. The samples were put in a -80ºC freezer. After 30 minutes, the samples were thawed and centrifuged for minutes at 14000 rpm at 4ºC. The relative light units (RLU) of the supernant were measured using the luminometer, and normalized to protein content using the BCA protein assay. Each RLU reading was obtained from four animals, and expressed as an average value. The guinea pigs without treatment were used as the control. 64 3.11.3 GFP expression in the cochlea of guinea pig 3.11.3.1 Complex preparation The micelles/DNA complexes were prepared by gently mixing 10 µL of micelle solution (1.35 mg/mL) with 1.0 µL of the sodium acetate/acetic acid buffer (0.02 M, pH 4.6) containing 2.5 µg GFP-plasmid. The solution was allowed to stand for 30 minutes before use. 3.11.3.2 Animal surgery and delivery of the complexes Albino guinea pigs weighing between 250 and 300 g were used for this study. The animals were initially anesthetized with a combination of ketamine (40mg/kg) and the analgesic xylazine (10mg/kg). The routine post-auricular approach was used to expose the tympanic bony bulla. A small opening was carefully made in the tympanic bulla with a pair of forceps to allow direct visualization of the round window membrane (RWM). A small piece of dry Gelfoam was placed in the groove, in direct contact with the round window membrane (RWM). An 11.0 µL of complex solution or naked DNA solution was injected into Gelfoam. This loading was to prevent the spread of the solution to neighboring tissues. The incision was closed in layers, and the total operating time was approximately 20 minutes. 3.11.3.3 Tissue processing Animals implanted with Gelfoam containing DNA complexes or naked DNA was sacrificed at days post surgery. The temporal bond was removed from both sides of the 65 head. Each bulla was opened using rongeurs to expose the cochlea. The stapes were removed and the cochlea was fixed by the injection of 4% paraformaldehyde through the RWM. The cochlea was then immersed in 4% paraformaldehyde at 4ºC overnight. After complete fixation, specimens were decalcified in 10% EDTA for days. After decalcification, the specimens were washed in PBS and dehydrated by immersion in increasing concentrations of alcohol, before being equilibrated in xylene. The specimens were then embedded in paraffin wax and sectioned radically at a thickness of µm on a Leica microtome (RM2125RT, Germany). 3.11.4 Luciferase expression in mouse breast tumor 3.11.4.1 Establishment of the tumor model Balb/C mice weighing between 20-30g were purchased from Singapore Animal Center and used for the in vivo study. 4T1 cells were maintained in RMPI 1640 medium supplemented with 10% FBS, mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C under an atmosphere with 5% CO2. To harvest 4T1 cells for mouse subcutaneous seeding, 4T1 cells were rinsed by PBS and detached by time trypsin. Thereafter, the medium containing cells were neutralized by RMPI 1640 medium with 10% FBS and centrifuged at 1000 rpm for minutes. The cells were then washed with PBS twice to remove FBS completely. The cells were dispersed evenly in free RMPI 1640 medium without FBS and injected subcutaneously beside the belly. The volume and density of cell injected per animal was 200 µl and 1×106 cells. After weeks, the tumor grew up to 200 mg. The tumor-bearing mice were available for in vivo studies. 66 3.11.4.2 Intratumor injection 30 µL of the blank micelles or paclitaxel loaded micelles complexes containing 2.0 µg DNA was injected into the tumor of each mouse. After 48 hours, the tumor was removed. The tumor samples were immersed in 1mL of the lysis buffer and then homogenized using a homogenizer (Heidolph, DIAX900, Germany). The samples were put in a -80ºC freezer. After 30 minutes, the samples were thawed and centrifuged for minutes at 14000 rpm at 4ºC and the supernant was collected. The relative light units (RLUs) of the solution were measured using a luminometer (Bio-Rad, U.S.A.), and normalized to protein content using the BCA protein assay (Bio-Rad, U.S.A.). Each RLU reading was obtained from eight animals, and expressed as an average value. The tumors (n=5) without treatment were used as the background. The background RLU was subtracted during the calculation of gene expression level. 3.11.4.3 Tail vein injection 200 µL of the complexes with different N/P ratios (n=5) containing 20 µg DNA was injected into the mouse through tail vein. After 48 hours, the tumor, heart, liver, spleen, lung and kidney of the mouse were harvested and processed by the similar protocol described in the previous section. The corresponding organs of the mice (n=5) without treatment were used as the background. 67 [...]... cut-off of 2 kDa and filtered by 0.45 µm filter The solution was added gradually into 2 mL of the sodium acetate/acetic acid buffer (0.02M, pH 4.6) containing 40 µg of DNA The mixture was then vortexed for 5 minutes before analysis of particle size and zeta potential 3. 6.2 Structural integrity of drug- loaded micelles after DNA binding It is known that the I3/I1 ratio from the emission spectra (λex =33 9... the control 64 3. 11 .3 GFP expression in the cochlea of guinea pig 3. 11 .3. 1 Complex preparation The micelles /DNA complexes were prepared by gently mixing 10 µL of micelle solution (1 .35 mg/mL) with 1.0 µL of the sodium acetate/acetic acid buffer (0.02 M, pH 4.6) containing 2.5 µg GFP-plasmid The solution was allowed to stand for 30 minutes before use 3. 11 .3. 2 Animal surgery and delivery of the complexes... spectra (λex =33 9 nm) of pyrene and the I 338 /I 333 ratio from the excitation spectra (λem =39 5nm) of pyrene change with changing the polarity of the microenvironment of pyrene [Jones M-C., 1999] When pyrene enters a more hydrophobic environment, the ratios increase To study the structural integrity of the pyrene-loaded micelles after DNA binding, the ratios were measured before and after DNA binding Freshly... different pH values and concentrations for 24 hours using the dialysis membrane with a molecular weight cut-off of 2 kDa The external aqueous phase was changed every hour The size and zeta potential of the drug- loaded polymeric micelles was analyzed as described in Section 3. 3.4 3. 4 .3 Determination of drug loading level and encapsulation efficiency The loading level and encapsulation efficiency of indomethacin,... processing 3. 10.5 In vitro synergistic effect of drug and gene 3. 10.5.1 In vitro synergistic effect of cyclosporin A and luciferase gene The synergistic effect of cyclosporin A and luciferase gene was performed against KB31-MA cell line, Cyclosporin A loaded polymeric micelles were prepared by dissolving 5mg of cyclosporin A and 15mg of P(MDS-co-CES) (polymer batch No 010704) into 5mL DMF and dialyzed... mixtures of 50μl of luciferase DNA (40 μg/ml) and 50 μl of ethidum bromide (0.8μg/ml) were added into 96well plate and allowed to incubate in room temperature for 30 mins first Fluorescence (λex =35 5nm, λem=590nm) of DNA/ ethidum bromide complexes solution was measured by fluorescence microplate reader (spectra MAX GEMXS, molecular devices, USA) and set as 100% of fluorescent intensity against 100 μl of naked... supplemented with 10% FBS, 2 mM of L-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37 °C under an atmosphere with 5% CO2 To subculture the confluent cells, the medium was removed first and washed with 5ml PBS buffer and then detached by 1ml trypsin 1/5 of the cells were passed to next flask 3. 9 Cytotoxicity of the micelles and the micelles /DNA complexes 3. 9.1 Material 3- (4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium... calculated as the ratio of loaded drug mass to the total mass of polymer and loaded drug 3. 5 Binding of DNA with blank and drug- loaded polymeric micelles 3. 5.1 Materials Agarose, biological grade, Bio-Rad, USA 52 Ethidium bromide, 10mg/ml, Sigma, USA DNA loading buffer, 5 times, Sigma, USA Tris-Acetic acid-EDTA Buffer Solution (TAE), 10 times, Sigma, USA, diluted to 1 times before using Sodium chloride,... N/P ratio of 15 The indomethacin-loaded micelles or the micelles /DNA complexes were then put in a dialysis membrane with a molecular weight cut-off of 2 kDa The dialysis membrane was placed in 50 mL of PBS (pH 7.4) at 37 °C At fixed time intervals, the external phase was sampled and analyzed for indomethacin level using the UV-VIS spectrometer at 31 8 nm 3. 8 Culture of cells 3. 8.1 Cell lines and materials... 3. 3.5 Stability of polymeric micelles The stability of polymeric micelles in de-ionized water, PBS, PBS containing 10% (v/v) fetal bovine serum or PBS containing 1% and 3% (wt) bovine serum albumin (BSA) was investigated by measuring size changes of the polymeric micelles as a function of time using the COULTER N4 Plus Particle Sizer 50 3. 4 Fabrication and characterization of drug- loaded micelles 3. 4.1 . 4.5 nm. The ratios of the peak intensities at 33 8 nm and 33 3 nm (I 33 8 /I 33 3 ) from the excitation spectra and I 3 (the third peak at 38 5nm)/I 1 (the first peak at 49 37 4nm) from the emission. emission spectra (λex =33 9 nm) of pyrene and the I 33 8 /I 33 3 ratio from the excitation spectra (λem =39 5nm) of pyrene change with changing the polarity of the microenvironment of pyrene [Jones M-C.,. was calculated as the ratio of loaded drug mass to the total mass of polymer and loaded drug. 3. 5 Binding of DNA with blank and drug- loaded polymeric micelles 3. 5.1 Materials Agarose, biological