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SIRIRAT CHOOSAKOONKRIANG,
1
BRIAN A. LOBO,
1
GARY S. KOE,
2
JANET G. KOE,
2
C. RUSSELL MIDDAUGH
1
1
The Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047
2
Valentis, Inc., Burlingame, California 94010
Received 22 October 2002; revised 24 February 2003; accepted 14 March 2003
ABSTRACT: The maingoal of this study was to determine the effects of polyethylenimine
(PEI) molecular weight and structure (750 kDa, 25 kDa, 2 kDa branched, and 25 kDa
linear PEI) and the nitrogen/phosphate (N/P) molar ratio on the physical properties and
transfection efciencies of PEI/DNAcomplexes. Fourier transforminfrared spectroscopy
revealed that DNA remained in the B conformation when complexed to all PEIs. Unique
alterations inthe circular dichroismspectraof DNAwere observedinthe presence of each
PEI, whereas differential scanning calorimetry measurements showed that all PEIs
examined destabilized supercoiled DNAat N/P<3/1, but not at higher ratios. Isothermal
titration calorimetry revealed the existence of protonation changes at low ionic strength
due to possible shifts in pK
a
of the ionizable groups of PEI during complex formation.
Twenty-ve kilodalton branched and 25 kDa linear PEI complexes showed the highest
transfection efciencies at an N/P ratio of 6:1 in COS-7 and CHO-K1 cells, respectively.
These investigations have detected alterations in the physical and colloidal properties of
the complexes that were sensitive to polymer structure, molecular weight, and polymer/
DNA ratio, but these properties did not directly correlate with their transfection
efciencies. To further probe any possible relationship between these parameters and
activity, a more rened biophysical analysis of any subpopulations in these samples that
may differ in transfection activity is suggested, although the existence of such species
remains unknown. 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm
Sci 92:17101722, 2003
Keywords: polyethylenimine; differential scanning calorimetry; isothermal titration
calorimetry; circular dichroism; DNA delivery; FTIR; gene delivery; physical character-
ization; plasmid DNA
INTRODUCTION
Gene therapy is a relatively new approach for the
treatment of genetic disorders using replacement
of a defective gene or introduction of new or
modied protein-based functions into cells to elicit
a therapeutic response. Development of efcient
and safe vehicles is one of the major obstacles to
the success of current gene delivery systems.
Gene transfer vectors currently available can be
broadly divided into two groups: viral and non-
viral. Although viral vectors have evolved to be
highly efcient gene delivery systems, nonviral
vectors have garnered considerable attention as
alternative vehicles because of their ease of syn-
thesis, unrestricted trans-gene size, low cost, and
low degree of immunogenicity.
1
Because naked plasmid DNA does not readily
penetrate most cellular membranes,
2
nonviral
gene delivery systems usually include agents to
increase gene delivery efciency. By complexation
to DNA, these agents typically form stable parti-
cles that are small enough to be readily trans-
ported and avoid rapid elimination by the
1710 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
Correspondence to: C. Russell Middaugh (Telephone: 785-
864-5813; Fax: 785-864-5814; E-mail: middaugh@ku.edu)
Journal of Pharmaceutical Sciences, Vol. 92, 17101722 (2003)
2003 Wiley-Liss, Inc. and the American Pharmacists Association
reticuloendothelial system. Upon cell entry, these
agents may also instill properties that promote
endosomal escape to avoid lysosomal degradation
and enhance nuclear entry.
One class of promising nonviral vector is the
polyethylenimines (PEI). PEI is a highly water-
soluble, positively charged, synthetic polymer in
which every third atom is a nitrogen that can be
protonated as well as provide a potential branch-
ing point. Approximately 20% of the nitrogens of
PEI are protonated under physiological condi-
tions.
3,4
As a result, the polymer can change its
ionization state over a broad pH range. The
cationic amines of both branched and linear PEI
also reduce the negative charge of DNA upon
complexation. This electrostatic interaction leads
to at least a partial condensation of the normally
large hydrodynamic volume of DNA.
5,6
This is seen
in 25 kDa PEI polyplexes as the formation of a
relatively homogenous population of toroidal par-
ticles of 4060 nm in diameter.
4
Branched and
linear PEIs of various molecular weights (MW) are
among the most transfection efcient nonviral
vectors in vitro
79
and in vivo.
2,1012
This high
transfection efciency is manifested in a variety of
target organs and delivery routes.
1317
The trans-
fection efciency of PEI depends on several factors
including polymer MW, the conformation of PEI
(e.g., linear vs. branched), and the target cell type.
This efciency may be further enhanced by the
ability of PEI to protect DNA from enzymatic
degradation.
15
Despite the potential use of PEI to deliver DNA,
the relationship between the properties of PEI/
DNA polyplexes and their ability to transfect
cells is poorly understood. Therefore, the purpose
of this study is to thoroughly characterize com-
plexes formed between DNA and various forms
of branched (2, 25, and 750 kDa) and linear PEI
(25 kDa) by a wide variety of biophysical methods.
We then use this information in an attempt to
correlate various physical properties with trans-
fection efciency in cell culture. To this end, the
secondary structure of DNA upon complexation
with PEI was investigated by using Fourier
transform infrared (FTIR) and circular dichroism
(CD) spectroscopy. The thermal stability of plas-
mid DNA complexed to PEI was studied using
differential scanning calorimetry (DSC). Addition-
ally, the enthalpy of binding between PEI and
DNA was explored using isothermal titration
calorimetry (ITC) and light scattering studies
were performed to assess the particle sizes and
zeta potentials of the complexes.
EXPERIMENTAL SECTION
Materials
Plasmid DNA pMB 290 (4.9 kbp), pMB 237
(9.1 kbp), and pMB 401 (encoding rey lucifer-
ase) [all >95% supercoiled (sc)] were provided by
Valentis, Inc. (Burlingame, CA). The DNA con-
centration was determined by UV absorbance
at 260 nm using an extinction coefcient of
0.02 (mg
1
cm
1
mL).
Branched PEIs (MW 750, 25, and 2 kDa) and
linear PEI (MW 25 kDa) were obtained from
Aldrich (Milwaukee, WI) and Polysciences, Inc.,
(Warrington, PA), respectively. The polymers
were used without further purication.
COS-7 and CHO-K1 cells were obtained from
American Type Culture Collection (Rockville,
MD). Dulbeccos modied Eagles medium, Hams
F-12 medium, and phosphate buffered saline
(PBS) were acquired from BioWhittaker (Walk-
ersville, MD). Opti-MEM (reduced-serum modi-
cation of MEM) and trypsin-EDTA were
purchased from Gibco (Greenland, NY). Fetal
bovine serum was obtained from Atlanta Biologi-
cals (Atlanta, GA). MTT [3-(4, 5-cimethylthiazol-
2-yl)-2, 5-diphenyl tetrazolium bromide] was
obtained from Sigma (St. Louis, MO).
All buffer materials (piperazine, MES, cacodylic
acid, phosphoric acid, BES, EDA, EPPS, PIPES,
ACES, boric acid, ethanolamine, HEPES, TES,
MOPS, TEA, and Tris) were obtained from Sigma
(St. Louis, MO). Nano-puried water was used to
prepare buffer solutions.
Preparation of Complexes
Stock PEI and DNA solutions were prepared
before each experiment at various molar ratios
of PEI nitrogen (N) to DNA phosphate (P) up to
N/P=10. The pH of the stock PEI solution was
adjusted to the desired pH using HCl. Complex
formation always utilized solutions of equal
volumes with the least concentrated component
being added to the more concentrated one.
Samples were continuously stirred during addi-
tion and equilibrated at room temperature for
~20 min before measurement. Complexes were
freshly prepared before each individual measure-
ment. Complexes were formed in 10 mM Tris
buffer, pH 7.4 unless otherwise noted.
FTIR Spectroscopy
FTIRspectra were obtained with a Nicolet Magna-
IR 560 spectrometer equipped with a mercury
PEI/DNA COMPLEXES 1711
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
cadmium telluride detector (Madison, WI). Sam-
ples were measured with attenuated total reec-
tance geometry where the sample in solution was
placed directly in the well of a ZnSe plate (effective
pathlength 12 mm). Spectra were obtained at
4-cm
1
resolution under a dry air purge by
accumulation of 256 interferograms. Subtraction
of the solvent (10 mM Tris buffer) was done using
the association peak of H
2
O near 2200 cm
1
as a
reference point.
18
Additional data analysis includ-
ed baseline correction (1804 to 904 cm
1
), seven
point Satvisky-Golay smoothing (if needed) and
Omnic Peaknd software. The nal DNA concen-
tration of all samples was 1 mg/mL whereas
the PEI concentration was varied for individual
complexes.
CD
CD spectra were obtained using a 0.1-cm path-
length rectangular cell at 258C, a Jasco J-720
spectropolarimeter (Easton, MD), and were cor-
rected by subtraction of buffer spectra. Spectra
were recorded from 350 to 200 nmat a scan rate of
20 nm/min and a resolution of 0.5 nm. Three
spectra were accumulated and averaged for each
sample. The nal DNA concentration of all
samples was 50 mg/mL (1.54 10
4
M DNA
bases). The CD signal was converted to molar
ellipticity [y], deg l mole
1
cm
1
, smoothed with
a Jasco Fast Fourier transform algorithm, and
then baseline adjusted to zero at 345 nmto correct
for a small contribution by differential light
scattering.
DSC
DSC was performed with a model 5100 Nano-DSC
[Calorimetry Sciences Corporation (CSC), Amer-
ican Fork, UT]. Measurements consisted of a
single scan from 0 to 1208C at 18C/min under
3 atm of pressure. All samples were prepared in
5 mMphosphate buffer, pH7.4 and were degassed
before measurement. Buffer exchange of DNA
was performed by dialysis using a Slide-a-lyzer
10,000 MWCO dialysis cassette (Pierce, Rockford,
IL) in 2.0 L buffer at 58C overnight. Baselines
were obtained by scanning with buffer in both the
sample and reference cells. Samples were ana-
lyzed in duplicate or triplicate. CpCalc software
(CSC) was used to subtract the baseline from the
sample thermogram. The data were converted to
molar heat capacity using the MW and concentra-
tion of DNA (0.5 mg/mL). All experiments used
pMB237 except for complexes of linear 25 kDa
PEI which used pMB290.
ITC
Calorimetric titrations of PEI into DNA were
conducted at 258C with a titration program of
25 injections of 10 mL using a CSC model 4200
isothermal titration calorimeter controlled by
ITCRun software (CSC). Titrations consisted of
an equilibration time of 300 s to establish a
baseline followed by injections at 5-min intervals.
Dialysis into the required buffers and degassing
were performed on all samples before use. DNA
and PEI concentrations were 0.31 and 5.45 mM,
respectively. The titration was stopped after
DNA/PEI aggregation occurred concurrent with
a loss of binding heat. Bindworks
TM
3.0 software
was used to integrate the raw data. Blank titra-
tions of PEI into buffer were performed and the
heats obtained were subtracted from each binding
titration of PEI into DNA. The observed enthalpy
of binding of PEI to DNA (DH
obs
) was calculated
from the average of the rst ve injections of the
binding titration, divided by the molar amount of
PEI added per injection.
pH Titrations of PEI
pH measurements of HCl titrations of PEI
solutions were performed using a Mettler Toledo
MP220 pHmeter (0.01 pHunit sensitivity) and an
accuT pH electrode. A water bath temperature
controller was used to maintain a sample tem-
perature of 25.08 0.18C. Titrations consisted of
incremental additions of 0.1 N HCl into 5 mL of
PEI solution (5.0 mg/mL) prepared in water. Each
titration was performed without prior pH adjust-
ment of the PEI solution.
Particle Size and Zeta-Potential Measurement
Samples were prepared in 10 mM Tris buffer pH
7.4, which was previously ltered through 0.2-mm
polysulfone lters (Gelman Science, MI). The
DNA concentration was held constant at 100 mg/
mL whereas the N/P ratios of the PEI/DNA com-
plexes were varied. Mean hydrodynamic dia-
meters were determined by cumulant analysis
using a dynamic light scattering (DLS) instru-
ment (BT 9000AT) equipped with a 50-mW HeNe
laser with a 532-nm emission wavelength (Broo-
khaven Instruments Corp., Holtsville, NY). Scat-
tered light was monitored at 908 to the incident
1712 CHOOSAKOONKRIANG ET AL.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
beam and the mean hydrodynamic diameter was
obtained from the diffusion coefcient using the
Stokes-Einstein equation. Three continuous mea-
surements of 1-min duration were taken for each
sample and the results averaged.
The zeta potentials of the PEI/DNA complexes
were determined by phase analysis light scatting
(PALS) at a scattering angle of 158 at 258C with a
Zeta PALS instrument (Brookhaven Instruments
Corp.). An electric eld strength between 14 and
16 V/cmwas used. Data were collected with 1015
cycles of the electric eld for each experiment and
averaged. The zeta potential was calculated from
the measured electrophoretic mobility using the
Smoluchowski approximation. The same samples
used for DLS measurements were used for zeta-
potential analysis.
Transfection Studies
Preparation of complexes was conducted as
described above, with the exception that a solu-
tion of plasmid pMB 401, encoding rey lucifer-
ase at 50 mg/mL was used. The resulting
complexes were diluted 10-fold into Opti-MEM
just before application to cells. All cells were
maintained in 75-cm
2
asks at 378C and 5% CO
2
with COS-7 cells grown in Dulbeccos modied
Eagles medium with L-glutamine and 4.5 g/L
glucose and CHO-K1 cells in Hams F-12 media
with L-glutamine, each supplemented with 10%
fetal bovine serum. Cells were subcultured every
4 days using standard procedures with trypsin/
EDTA for cell lifting. Before seeding, the cells
were trypsinized, counted, and diluted to a
concentration of approximately 80,000 cells/mL.
Then 0.1 mL of this dilution was added to each
well of a 96-well plate and the cells were
incubated in a humid 5% CO
2
incubator at 378C
for 1820 h. Immediately before transfection, the
cells were washed once with PBS and the complex
(250 ng of DNA) was added to each well. Cells
were incubated with the complexes for 5 h. The
transfection agent was then removed and 100 mL
of culture medium was added followed by a
further incubation of 48 h. A luciferase expression
assay was performed using the Luciferase Assay
System
TM
from Promega (Madison, WI) following
the manufacturers recommended protocol. The
cells were washed with PBS and 20 mL of 1X lysis
buffer was added per well. The cells were allowed
to stand at roomtemperature for 30 min. The plate
was then placed into a Fluostar
TM
Galaxy micro-
titer plate reader (BMG, Germany) equipped with
an injector. A volume of 100 mL of luciferase assay
reagent was added to each well immediately
before measurement. A luciferase standard cali-
bration curve was obtained and used to convert
light units to nanograms of luciferase. The data
are reported as the meanstandard error for a
minimum of three to ve samples per data point.
Assessment of Cytotoxicity (MTT Assay)
Both COS-7 and CHO-K1 cells were grown as
described in the transfection experiments. Cells
were treated with the PEI/DNA complexes (using
the same N/P ratio used in the transfection
studies) or PEI alone (using the same concentra-
tion of PEI present in the complexes) for 5 h. The
complexes or PEI were then removed and 100 mL
of culture medium was added as needed for each
cell type. The cells were then incubated for an
additional 40 h at 378C. At this point, 11 mL of
MTT was added to each well and the cells were
incubated at 378C with 5% CO
2
for 3 h. An aliquot
of 110 mL of MTTSS (10% Triton X-100 0.1 N
HCl in 125 mL of isopropanol) was added to each
well and the cells were incubated at 378C over-
night. The absorbance at 570 nm was measured
using a Fluorostar
TM
microtiter plate reader and
the percent cell viability was calculated using the
following equation:
% Cell viability
=
[Absorbance of the test sample + 100[
[Absorbance of control (cells alone)[
(1)
RESULTS
Infrared Spectral Properties of PEI/DNA Complexes
In Figure 1A, representative infrared spectra of
DNA and its complexes with 750 kDa PEI are
shown stacked in order of increasing N/P ratio,
with uncomplexed DNA at the bottom and
PEI alone at the top. In the absence of polymer,
DNA is in the B conformation as indicated by
the presence of the guanine/thymidine (G/T)
carbonyl stretching band at 1714 cm
1
(represen-
tative of interstrand base-pairing), an asymmetric
phosphate stretching vibration at 1224 cm
1
,
and a sugar-phosphate coupled vibration at
970 cm
1
.
19,20
Additional bands near 1328, 1281,
and 897 cm
1
(the latter not shown) support
this assignment of the B conformation.
21,22
DNA
vibrational bands arising from base carbonyls
PEI/DNA COMPLEXES 1713
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003
(1715 cm
1
), the imidazole nitrogen of guanine
(1492 cm
1
), and the phosphate backbone
(1224 cm
1
) have been found to be particularly
sensitive to changes in cationic lipid/DNA com-
plexes.
19,23
Interference from PEI CH
2
scissoring
(1471 cm
1
) and NH bending (1515 cm
1
)
vibrations, however, make it difcult to consis-
tently resolve the 1492 cm
1
guanine imidazole
nitrogen mode of DNA. Therefore, only the base
carbonyl and antisymmetric phosphate stretching
vibrations were monitored in this study.
Discrete changes in these two vibrational
frequencies upon addition of PEI to DNA are
shown in Figure 1BI. The frequency of the base
in-plane C