Novel biodegradable cationic core shell nanoparticles for codelivery of drug and DNA 3

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 Hexa

Chapter 3

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

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

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, 3 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 3 times with

20 mL of 1 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 5 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 methyldietheneamine sebacate) (PMDS) and

poly(N-methyldietheneamine 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 1 hour later, and the reaction was allowed to proceed at room temperature for 3 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 dissolved in 150 mL

of toluene and washed three times with 45 mL of NaCl-saturated aqueous solution, pH of which was adjusted to ~ 8 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

2 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 4 more times The yield was 30% to 70%

Poly{(N-methyldietheneamine adipate)-co-[(cholesteryl oxocarbonylamido ethyl)

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 1 hour later, and the reaction was allowed to proceed at room temperature for 3 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 8 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

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

N-(2-bromoethyl)carbarmoyl cholesterol(Be-chol)

Scheme 1 Synthesis of N-(2-bromoethyl)carbarmoyl cholesterol (Be-chol)

P(MDS-co-CES)(n=8) P(MDA-co-CEA)(n=4)

sebacoyl chloride(n=8)

adipoyl chloride (n=4)

CH 3

NH O

H H

H O

CH3(CH 2 ) 2 (CH 2 ) 2

(CH2)2(CH 2 ) 2

O

C

O

triethylamine OH

N HO

+ -Cl

O C

O

m

CH3(CH2)n

(CH2)n

(CH2)2 (CH2)2

(CH2)2(CH2)2

CH3N O

O (CH2)2 N (CH 2 ) 2

p

O C

O NH

(CH 2 ) n

C O

CH 3

(CH 2 ) 2 (CH 2 )2(CH2)n

q

O C

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 1 time

3.2.4 Gel permeation chromatography (GPC) analysis

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 5 mL of THF and the solution was then filtered The mobile phase was THF with a flow rate of 1 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

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 2 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 5 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 4 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

(I 338 /I 333 ) from the excitation spectra and I 3 (the third peak at 385nm)/I 1 (the first peak at

374nm) from the emission spectra were analyzed as a function of polymer concentration The CMC value was taken from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at the low concentrations

3.3 Fabrication and characterization of polymeric micelles

3.3.1 Materials

N,N-Dimethylformamide (DMF), ACS grade, Merck, USA

Sodium acetate, ACS grade Merck, USA

Acetic acid, ACS grade, Merck, USA

Sodium acetate/Acetic acid buffer, 0.2M, 0.02M, 0.01M, pH 4.6, pH5.6, self-prepared Dialysis membrane, MWCO 2000, Sigma, D-7884 or Spectrum/pro, USA

Phosphotungstic acid, ACS grade, Sigma, USA

Carbon coated TEM grid, SPI, USA

Fetal bovine serum, Sigma, USA

Bovine serum albumin (BSA), Sigma, USA

3.3.2 Preparation of polymeric micelles

Polymeric micelles were prepared by a membrane dialysis method using the cationic polymers Briefly, a certain weight of polymer was dissolved in 5 mL of DMF, which was then dialyzed against 500 mL of de-ionized water or sodium acetate/acetic acid buffer with different concentrations and pH values for 48 hours using a dialysis membrane with a molecular weight cut-off of 2 kDa (Sigma, D-7884 or Spectrum 2000)

The external aqueous solution was changed every hour for the first 8 hours and then

every 8 hours

3.3.3 Transmission electron microscopy (TEM) measurements

A drop of the solution containing freshly prepared micelles and 0.01% (w/v) phosphotungstic acid was placed on a copper grid coated with carbon, which was air-dried TEM studies were performed on a Philips CM300 microscope (Netherlands) with

an electron kinetic energy of 300 keV

3.3.4 Particle size and zeta potential measurements

The particle size and zeta potential of the freshly prepared micelles were measured by Zetasizer equipped with a He-Ne laser beam at 658 nm (scattering angle: 90°) (3000HS, Malvern Instrument Ltd., UK) or COULTER N4 Plus Particle Sizer with a He-Ne laser beam at 632.8 nm (scattering angle: 90°) or ZetaPals equipped with a He-Ne laser beam

at 658 nm (scattering angle: 90°) (Brookhaven Instruments Corp, USA) at 25ºC Each measurement was repeated 5 times An average value was obtained from the five measurements

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

3.4 Fabrication and characterization of drug-loaded micelles

3.4.1 Materials

Indomethacin, >99%, Sigma, USA

Paclitaxel, >99%, LC laboratories, USA

Cyclosporin A (CyA), >99%, LC laboratories, USA

D,L-Verapamil hydrochloride, Sigma, USA

3.4.2 Preparation of drug-loaded micelles

Indomethacin, pyrene, paclitaxel, cyclosporin A, D,L-verapamil were used as model drug compounds Drug-loaded micelles were prepared by the membrane dialysis method Briefly, a certain amount of drug and polymer was dissolved in 5mL of DMF The mixture was dialyzed against 500 mL of de-ionized water or sodium acetate/acetic acid buffer with 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, pyrene, paclitaxel, verapamil was determined using a UV-VIS spectrometer (Shimadzu UV-2501, Shimadzu, Japan) Briefly, for the indomethacin-loaded micelles, a fixed mass of freeze-dried micelles was dissolved in DMF The solution was measured directly by the UV-VIS

spectrometer For the pyrene, paclitaxel and verapamil-loaded micelles, 100 µL of pyrene-loaded micelle solution was mixed with 2 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 5 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 1 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