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Ronny Martien
Brigitta Loretz
A
...
Schnurch
¨
Department of Pharmaceutical
Technology,
Institute of Pharmacy,
Leopold-FranzensUniversity of Innsbruck,
Innrain 52, 6020 Innsbruck,
Austria

Oral Gene Delivery: Design
of Polymeric Carrier Systems
Shielding toward Intestinal
Enzymatic Attack

Received 5 January 2006;
revised 23 March 2006;
accepted 30 March 2006
Published online 11 April 2006 in Wiley InterScience (www
...
wiley
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DOI 10
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20521

Abstract: The gastrointestinal tract poses a variety of morphological and physiological barriers
to the expression of target genes
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Within this study, a chitosan–enzyme inhibitor conjugate has been generated and characterized
...
The
enzyme inhibitor aurintricarboxylic acid (ATA) was covalently bound to chitosan to improve the enzymatic stability of nanoparticles formed with this polymer and pDNA
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5 6 26 nm and a zeta potential of À13
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24 mV (n ¼ 3–4)
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05) more stable than
unmodified chitosan/pDNA nanoparticles
...
6-fold higher transfection rate than chitosan/pDNA nanoparticles in
the Caco-2 cell line, thus creating a promising carrier for orally administered therapeutic
genes
...
Biopolymers 83: 327–336, 2006
This article was originally published online as an accepted preprint
...
You can request a copy of the preprint by emailing the
Biopolymers editorial office at biopolymers@wiley
...
1 The
concept, however, is so far strongly limited by an insufficient access of the target tissue
...
2 The gastrointestinal tract, however, sets a variety
of morphological and physiological barriers that can
limit internal absorption of the therapeutic gene
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Bernkop Schnurch; e-mail: andreas
...
ac
...
martien@uibk
...
at
Biopolymers, Vol
...


327

328

Martien, Loretz, and Schn€rch
u

coacervation between cationic polymeric excipients
and nucleic acid
...
5,6 Chitosan, a polycationic polymer comprising D-glucosamine and
N-acetyl-D-glucosamine linked by (14)-glycosidic
bonds, is nontoxic, biocompatible, biodegradable, acid
soluble, and positively charged
...
8 Additionally, it can
be easily combined with auxiliary agents, such as
enzyme inhibitors,9 which can protect DNA inside particles toward degradation by nucleases
...
Aurintricarboxylic acid (ATA) was chosen
as the inhibitory agent, as it has already been used as
nuclease inhibitor in the GI tract10 to enhance the
transfection rate in gene delivery
...
It
was furthermore the aim of this study to evaluate the
potential of this delivery system to provide protection
against salts and enzymes, such as lysozyme and
DNase, being present in the intestinal fluid
...


MATERIALS AND METHODS
Materials
Low viscous chitosan [2-amino-2-deoxy-(1?4)--D-glucopyranan] was obtained from Fluka Co
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, Seelze, Germany
...
, CA, USA)
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WernerFelmeyer, Institute of Medical Chemistry and Biochemistry, University of Innsbruck (Innsbruck, Austria)
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Restriction endonuclease EcoRI, 1 kb DNA ladder,
and orange-blue loading dye were obtained from Promega
...


Presumptive substructure of chitosan-ATA

SERVA Electrophoresis GmbH (Heidelberg, Germany)
...


Small Intestinal Fluid
The small intestinal fluid from a freshly slaughtered pig
was collected and frozen on dry ice
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Samples were purified by filtration through gases and centrifugation at 4500 rpm for
15 min to remove undigested nutrient residues
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Cell Line
Caco-2 cells were obtained from the laboratory of Prof
...

W
...
Fetal
minimum essential medium (MEM), NaHCO3, trypsin
(0
...
Fetal calf
serum was obtained from Gibco Laboratories, Lenexa, KS,
USA
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3 kb) was used to monitor protection of chitosan–ATA
complexes against endonuclease
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coli XL-1 and isolated and purified using an
alkaline lysis method
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The pDNA concentration was determined
by measuring absorbance at 260 nm utilizing an UV–vis
spectrophotometer (UV-1202, Shimadzu Co
...

Purity was confirmed by enzyme digestion with EcoRI followed by gel electrophoresis with 0
...
The concentration of DNA solution was adjusted to 50 g/mL
Biopolymers DOI 10
...
The standardized pDNA solution was stored at
À208C until further use
...
The reaction mixture was stirred for 1 h
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0 with 4 M NaOH
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4 with 70% methanol under reduced pressure and
followed by dialyzation against 4 L of distilled water for
2 days
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Depolymerization of chitosan with NaNO2 can reduce molecular mass of low viscous
chitosan from 213 to 100 kDa
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1% (v/v) acetic acid
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5, 1:5, and 1:10,
respectively
...
0 by the addition of
5 M NaOH
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1 M and the reaction mixtures were incubated at room
temperature under permanent stirring for 12 h
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025 M NaOH, and once more
two times against distilled water
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329

Preparation of Chitosan–ATA
Nanoparticles
Chitosan–ATA nanoparticles were generated by complex
coacervation of chitosan–ATA and pDNA
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02% unmodified chitosan solution in a 5 mM acetate
buffer (pH 5
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04% chitosan–ATA conjugate solutions (weight ratio 1:2
...
0) was heated to
508C for 10 min
...
0
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Thereafter, 400 L of unmodified chitosan solution was added while vortexing at 2500 rpm for 20 s
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For control,
unmodified chitosan nanoparticles were synthesized as
described previously
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The size of DNA nanoparticles was determined by photon correlation spectroscopy at
238C using a PSS Nicomp 380 ZLS particles sizer (Nicomp
Laboratory Instrument, Santa Barbara, CA, USA) with laser
wavelength 650 nm at a 908 angle
...
16 The size
was calculated assuming solid particles, and the number of
weighting distribution was fit to a Nicomp size distribution
curve which in the ZPW388 v 1
...
Based on Nicomp Distribution
Analysis, the number of weighting distribution measured
every particle in the solution
...


Determination of Degree of Modification
The amount of immobilized ATA was determined by measuring absorbance of the conjugates at 420 nm (Spectrophotometer DU1 Series 600, Beckman Instruments, Inc, CA,
USA) in distilled water
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Fluorescence Binding Titration
DNA condensation was measured by quenching ethidium
bromide fluorescence as described previously
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0, containing 1 M EtBr
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The fluorescence was measured at 544/590 nm (exication/emission) in FLUOstar* Galaxy, BMG Labtechnology,
Offenburg, Germany
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1002/bip

Zeta Potential Measurement
...
78, dielectric constant of water 78
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933 cP, and E-fields strength 2 V/cm
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65 software
from NICOMP Particles Sizing Systems
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Drops of freshly 10 L prepared nanoparticle solution
were placed on pioloform-coated grids and air dried for
15 min
...
These grids
were examined with a ZEISS LIBRA1 120 transmission
¨
electron microscope (Carl Zeiss A6, Gottingen, Germany)
with an in-column energy filter (EFTEM)
...
Thus, nanoparticles were analyzed at 0, or 50 to
70 eV energy loss, depending on their density
...

micrographs were obtained from a ProScan Slow Scan
CCD camera system using iTEM1 5
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Proscan, Electronische System
GmbH, Lagerlechfeld, Germany)
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To evaluate
the stability of unmodified chitosan/pDNA and chitosan–
ATA/pDNA nanoparticles against the salts solution, they
were incubated with artificial intestinal fluid
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0 with acetic acid
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After 4 h incubation, each particle suspension was investigated via agarose gel electrophoresis
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7% (w/v) gel
for 2 h at 60 V using ethidium bromide for visualization
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The gel slices were weighted and melted at 998C
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The
amount of particles left was determined by fluorescent measurement with ethidium bromide staining DNA at 544/590
nm (exication/emission) by FLUOstar* Galaxy, BMG Labtechnology
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The stability of nanoparticles
was analyzed after their incubation at 378C in lysozyme solution (0
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18 This test was per-

formed by adding 6
...
After 4 h of incubation, 10 L of trypsin solution (50 mg/mL) was added to
stop the reaction
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Protection against DNase I Degradation
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0), 100 mM MgSO4, and 10 mM CaCl2) at 378C for
1, 2, and 4 h
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0,
solutions was added to each sample to stop the reaction
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Degradation of particles was investigated by 0
...

Incubation with Intestinal Juice
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Thereafter, 10 L of 5 mM EDTA, pH 8
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The degree of nanoparticle degradation was investigated via agarose gel electrophoresis as described above
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Caco-2 cells were maintained at 378C under 5% CO2 and
90% relative humidity in MEM supplemented with 20%
(v/v) fetal calf serum, 2
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1002/bip

Oral Gene Delivery

331

Table I The Amount of Immobilized ATA

Conjugates
Chitosan:ATA 1:10
Chitosan:ATA 1:5
Chitosan:ATA 1:2
...
01
0
...
01
0
...
1029
0
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0258
0
...
92
9
...
92
0
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4 6 2
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9 6 4
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8 6 1
...
00 6 0

Values are means 6 SD of three experiments
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Cells were seeded in 12-well plates (5 Â 104 cells/
well) and grown in MEM medium at standard culture condition for 24 h
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Unmodified chitosan/
pDNA nanoparticles served as control
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Culture cells were harvested for green fluorescent protein
assay after 36 h incubation
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5, 0
...
One hundred microliters of
each cell lysate was transferred to a 96-well plate
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Statistical Data Analyses
Green Fluorescence Protein Assay
Culture media were discarded and the cells were washed
with PBS, pH 7
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Cells were detached with trypsin, suspended in PBS, and collected by centrifugation
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05 as the level of significance
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5 for Windows
(The MathWorks, Inc
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FIGURE 3 Titration of DNA in the present of ethidium bromide by polymers
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Chitosan–ATA 1:2
...
Chitosan–ATA 1:5 conjugate (~)
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Indicated values are means 6 SD of three experiments
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1002/bip

332

Martien, Loretz, and Schn€rch
u

RESULTS AND DISCUSSION
Conjugation of Chitosan–ATA
The covalent coupling of ATA to chitosan was achieved by formation of an amide bond between the carboxylic acid group of the complexing agent and the primary amino group of the polymer (Figures 1 and 2)
...
The amount of primary amino
groups remaining after coupling reaction is crucial for
the formation of nanoparticles with DNA
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Development and Characterization
of Nanoparticles
The complex formation between plasmid DNA
(pEGFP) and the chitosan–ATA conjugate is initially
electrostatic by the attraction between the anionic
DNA and the cationic carrier material
...
Dynamic light scattering
measurements showed that the zeta potential of
unmodified chitosan/pDNA nanoparticles is 5
...
38 mV and that of the chitosan–ATA/pDNA nanoparticles is approximately À13
...
24 mV
...
19 The negative charge of the chitosan–ATA
nanoparticles is primarily based on pDNA but to
some extent also is based on ATA being covalently
attached to chitosan
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20 Electrostatic repulsions which must be overcome by high salt
concentrations or by the correlated fluctuations of territorially bound multivalent cation and repulsive excluded volume interactions with DNA in the solution
...
e
...
22

FIGURE 4 Nicomp nanoparticles size distribution
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4 6
26 nm (n ¼ 3)
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5626 nm (n ¼ 3)
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5 showed the
principal ability to form particles, even when 23
...
The more ATA was bound, the less
primary amino groups remained on the polymer
...
The
displacement of EtBr from DNA was monitored by
measuring the ethidium bromide fluorescence as cationic polymer added to solution
...
Fewer cationic
charges remaining, however, reduce in turn the capability of conjugates to form complexes with pDNA
and the particles nevertheless formed are comparatively smaller
...
For this reason a mixture of unmodified
Biopolymers DOI 10
...
Image of colony chitosan–
ATA/pDNA nanoparticle in solution (A) and a single chitosan–ATA/pDNA nanoparticle (B) with
12,500Â magnification, 50 eV energy loss
...
In Figure 4, the particle size of chitosan–
ATA/pDNA nanoparticles is shown to be 98
...
4 6 26 nm
...

Results showed that the shape of the nanoparticles is
fairly spherical (Figure 5)
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complex
...
In general, the polyelectrolyte
complex formed with higher ionic strength is less stable than those with lower ionic strength,25 because
salt usually has a shielding effect on the electrostatic
force, and hence weakens the salt bond between the
complexes
...
As shown for
several other polycations, the interaction between
chitosan and DNA is electrostatic
...
24 The percentage of particles
remaining was slightly reduced upon the incubation
with AIF
...
05) as shown in Figure 6
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1002/bip

Chitosan is a linear polysaccharide of both N-acetylglucosamine and glucosamine units randomly distributed
...

The rate of biodegradation of chitosan by lysozyme
was found to be dependent on the DA and water solubility
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27 Lysozyme hydrolyzes the glycosidic bond of the
biopolymer and is present in gastrointestinal tract
...
The concentration of lysozyme in the
gastric juice is 43–106 g/mL
...
Indicated values are means 6 SD of three experiments
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The degradation of the particles
can be attributed to the hydrolysis of chitosan substructures being exposed on the surface of particles
by lysozyme
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18 ATA being bound to the polymer has an inhibitory effect on lysozyme (Figure 7)
...


FIGURE 7 Stability of chitosan/pDNA nanoparticles
(n) and chitosan–ATA/pDNA nanoparticles (~) in lysozyme (0
...
Indicated values are means 6
SD of three experiments
...
05
...
On the other
hand, chitosan/pDNA nanoparticles were completely
digested within 4 h of incubation with an equal
amount of DNase I
...

This in vitro experiment demonstrates that ATA is
indeed capable of inhibiting the nuclease activity in
the small intestinal fluid
...
30
Chitosan–ATA conjugate increased encapsulated
DNA stability due to the ATA-mediated inhibition of
nuclease activity
...
31 The stability of nanoparticles in
the intestinal fluid however is a key issue in oral gene
delivery
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Intestinal fluid contains
mainly cation-dependent nucleases, such as DNase I,
which can be inhibited by ATA
...
The results from intes-

FIGURE 8 Agarose gel electrophoresis for DNase I protection assay
...
Lane 1, untreated control DNA; lane 2, DNA after incubation for 1 h;
lane 3, untreated control chitosan/pDNA nanoparticles;
lanes 4 and 5, chitosan/pDNA nanoparticles after 2- and 4-h
incubation with DNase I; lane 6, untreated control chitosan–ATA/pDNA nanoparticles; lanes 7 and 8, chitosan–
ATA/pDNA nanoparticles after 2- and 4-h incubation with
DNase I
...
1002/bip

Oral Gene Delivery

FIGURE 9 Stability of chitosan/pDNA nanoparticles
(n) and chitosan–ATA/pDNA nanoparticles (~) in intestinal fluid at 378C
...
*Differ from chitosan–pDNA nanoparticles,
p < 0
...


tinal fluid stability test are shown in Figure 9
...
Covalent coupling of
ATA to chitosan backbone was chosen to provide
more protection of pDNA against degradation by endonucleases
...
In contrast, degradation assay with chitosan–ATA/pDNA nanoparticles showed that about
60% of particles remain stable (Figure 9)
...


335

was encapsulated in chitosan–ATA conjugate, led to
2
...
The transfection rate with nanoparticles is also
size dependent
...
Chitosan–ATA/pDNA
nanoparticles have a smaller particle size than chitosan/pDNA (Figure 4)
...
36 As chitosan/pDNA nanoparticles
exhibit a more positive zeta potential, the attachment
of particles to the cell membrane is less pronounced
than that of chitosan–ATA/pDNA nanoparticles
...
It has been
shown in Figures 7 and 8 that chitosan–ATA conjugates protect DNA against enzymatic degradation
...
In the culture
media, nuclease activity comes from heated-inactivated FCS (data not shown)
...
In the cytoplasm, chitosan–ATA nanoparticles are likely not destroyed38 and directly move to
the nuclear pore complex
...
Thereby it is important to assess the effectiveness of uptake of the carrier and associated gene
cargo into the target cell
...
34 The human colon adenocarcinoma cell line (Caco-2), which was used for these
studies, has features similar to the absorptive intestinal cell, such as microvilli
...
11 Figure 10 demonstrated that chitosan–ATA/pDNA nanoparticles let to
a higher transfection rate in the Caco-2 cell than
unmodified chitosan/pDNA
...
1002/bip

FIGURE 10 Expression of green fluorescence protein in
Caco-2 cell after transfection with nanoparticles
...


336

Martien, Loretz, and Schn€rch
u

CONCLUSION
A new type of conjugate from chitosan–ATA was
synthesized and used to encapsulate pDNA
...
Transfection studies with
Caco-2 cell line showed that chitosan–ATA nanoparticles lead to a higher transfection rate than chitosan/
pDNA nanoparticles
...

This work was supported by a scholarship from the Austrian Federal Ministry for Education, Science, and Culture
to RM
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Ozbas-Turan, S
...
; Kabasakal, L
...
; Akbuga, J
...

2
...
; Ochiya, T
...

3
...
; Borchard, G
...
C
...
;
Junginger, H
...
Int J Pharm 2005, 299, 155–166
...
Illum, L
...
; Hinchcliffe, M
...
N
...
S
...

5
...
; Alonso, M
...
; Torres, D
...

6
...
; Andrieux, K
...
J
...
; Gursoy, R
...
; Couvreur, P
...
Int J Pharm 2005,
298, 378–383
...
Pouton, C
...
; Seymour, L
...
Adv Drug Deliv Rev
1998, 34, 3–19
...
Janes, K
...
; Calvo, P
...
J
...

9
...
J Controlled Release 1998, 52,
1–16
...
Chen, C
...
; Chao, Y
...
H
...
J
...
W
...

11
...
; Steenland, P
...
; McDonald, R
...
;
Sanchez, R
...
; Watts, T
...
; Zabner, J
...
W
...

12
...
Biochim Biophys Acta 1966, 119, 198–
200
...
Huang, M
...
; Lim, L
...
Pharm Res 2004, 21,
344–353
...
Tang, M
...
; Szoka, F
...
Gene Ther 1997, 4, 823–
832
...
Leong, K
...
; Mao, H
...
; Truong-Le, V
...
; Roy, K
...
M
...
T
...


16
...
; Sanchez, A
...
; Calvo, P
...
J
...

17
...
-G
...
Wiss Tabellen Geigy, 2005
...
de Campos, A
...
; Diebold, Y
...
L
...
;
Sanchez, A
...
J
...

19
...
C
...
A
...
; Liu, G
...
; Harpst, J
...
; Hanson, R
...
J Biol
Chem 1997, 272, 7398–7407
...
Marty, J
...
; Oppenheim, R
...
; Speiser, P
...

21
...
A
...

22
...
J
...

23
...
Q
...
; Troung-Le, V
...
; Janes, K
...
;
Lin, K
...
; Wang, Y
...
T
...
W
...

24
...
; Mao, H
...
; Huang, S
...
; Leong, K
...
Nat
Med 1999, 5, 387–391
...
Chansiri, G
...
T
...
V
...
L
...

26
...
; Zhang, S
...
; Zhang, L
...
; Xiong, C
...
;
Peng, Y
...
J Biomed Mater Res Part B Appl Biomater
2005, 72B, 299–304
...
Don, T
...
; Hsu, S
...
; Chiu, W
...
J Polym Sci Part A
Polym Chem 2001, 39, 1646–1655
...
http://www
...
com
...
asp
29
...
; Sanchez, A
...
; Soriano, I
...
; Vila-Jato, J
...
; Alonso, M
...
Colloids Surf B Biointerfaces 2000, 18, 315–323
...
Glasspool-Malone, J
...
; Drabick, J
...
;
Malone, R
...
Mol Ther 2000, 2, 140–146
...
Barry, M
...
; Pinto-Gonzalez, D
...
M
...
J
...
R
...
A
...

32
...
; Stein, U
...
; Fichtner, I
...
M
...

33
...
F
...
Biochem Biophys Res
Commun Biochim Biol Cell 2000, 78, 405–414
...
Ma, Z
...
; Lim, L
...
Pharm Res 2003, 20, 1812–1819
...
Desai, M
...
; Labhasetwar, V
...
; Levy, R
...
;
Amidon, G
...
Pharm Res 1997, 14, 1568–1573
...
Ueyama, T
...
; Kami, K
...
; Kobayashi, T
...
; Lennartz, M
...
; Takeya, R
...
; Saito, N
...

37
...
; Cuie, Y
...
; Shi, Q
...
; Benderdour, M
...
; Fernandes, J
...

Biomaterials 2006, 27, 2060–2065
...
Escriou, V
...
; Scherman, D
...
Adv
Drug Deliv Rev 2003, 55, 295–306
...
Hebert, E
...


Reviewing Editor: C
...
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