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

39


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

40

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.

41

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 (R
f
)
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(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

42

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 NaCO

3
. 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)

43

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.


44

3.1.6 Synthesis of PEGylated P(MDS-co-CES)
PEGylated PMDS was first characterized by using
1
H-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
1
H-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.
BrCH
2
CH
2
NH
2
NH
Br
O
H
H
H
O
CH
3
CH
3
H
3
C
H
3
C
H
3
C
O

H
H
H
O
CH
3
CH
3
H
3
C
H
3
C
H
3
C
Cl
+
triethylamine
1
bromoethylamine
cholesteryl Chloroformate
N-(2-bromoethyl)carbarmoyl cholesterol(Be-chol)

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


45

P(MDS-co-CES)(n=8)
P(MDA-co-CEA)(n=4)
Be-chol
O
C
O
C
O
q
(CH
2
)
n
(CH
2
)
2
(CH
2
)
2
CH
3
N
O
O
C
(CH
2
)

n
poly(N-methyldietheneamine sebacate)(PMDS)(n=8)
poly(N-methyldietheneamine adipate)(PMDA)(n=4)
N-methyldiethanolamine
+
Br
sebacoyl chloride(n=8)
adipoyl chloride (n=4)
CH
3
NH
O
H
H
H
O
CH
3
CH
3
H
3
C
H
3
C
H
3
C
O

C
O
p
(CH
2
)
2
(CH
2
)
2
N
O
O
N
CH
3
(CH
2
)
2
(CH
2
)
2
(CH
2
)
2
(CH

2
)
2
(CH
2
)
n
(CH
2
)
n
CH
3
m
O
C
O
C
O
triethylamine
OH
N
HO
+
-Cl
O
C
O
Cl-C
2

3

Scheme 2 Synthesis of P(MDS-co-CES) and P(MDA-co-CEA).

Cl-C
O
C
O
-Cl
+
HO
N
OH
triethylamine
O
C
O
C
O
m
CH
3
(CH
2
)
n
(CH
2
)
n

(CH
2
)
2
(CH
2
)
2
(CH
2
)
2
(CH
2
)
2
CH
3
N
O
O
N
(CH
2
)
2
(CH
2
)
2

p
O
C
O
H
3
C
H
3
C
H
3
C
O
H
H
H
O
NH
CH
3
sebacoyl chloride(n=8)
adipoyl chloride (n=4)
Br
+
N-methyldiethanolamine
poly(N-methyldietheneamine sebacate)(PMDS)(n=8)
poly(N-methyldietheneamine adipate)(PMDA)(n=4)
(CH
2

)
n
C
O
O
N
CH
3
(CH
2
)
2
(CH
2
)
2
(CH
2
)
n
q
O
C
O
C
O
Be-chol
Pegylated P(MDS- co-CES)(n=8)
Pegylated P(MDA-co-CEA)(n=4)
mPEG

+ CH
3
-(OCH
2
CH
2
)
S
-OH
2
3
CH
3
-(OCH
2
CH
2
)
S
-O
(CH
2
CH
2
O)
S
-CH
3
CH
3

CH
3

Scheme 3 Pegylation of P(MDS-co-CES) and P(MDA-co-CEA).

46


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

3.2.2
1
H-NMR analysis
The
1
H-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

47

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 (T
g
) 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 (T
g
) 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

48

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

49

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).

50

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.

51

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

52

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 SymmetryShield
TM
C
8
4.6×15.0 cm
column fitted with a C
8
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 r
2

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

53

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, 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. 8 µL) by using the same buffer employed for preparation of the
micelles. 2 µL of 5 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

54

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

55

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 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 I
3
/I
1
ratio from the emission spectra (λex=339 nm) of pyrene and
the I
338
/I
333
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.

56

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 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 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

57

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 1 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, 2 mM of L-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin at

37°C under an atmosphere with 5% CO
2
using 75ml plastic flask. 4T1 cells were
maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640) 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% CO
2
. 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

58

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% CO
2
at 37°C for a certain period. Each sample was
tested in 8 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% CO
2
at 37°C for 3 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.


59

3.9.3 Micelles/DNA complexes
The cytotoxicity of the micelles/DNA complexes at the N/P ratios of 5 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

60

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 5 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 8 hours and then every 8 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.

61


3.10.3 In vitro luciferase expression
The cells were seeded onto 24-well plates at a density of 8×10
4
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 luciferase-
encoded plasmids were added to each well. After 4 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 phosphate-
buffered 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 5 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 2×10
5
cells per well, and cultivated in 1 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 4 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

62

with 1.0 mL of PBS. 0.3 mL of 1 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 5 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 KB-
31-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

63

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