GENE DELIVERY

Journal of Controlled Release 130 (2008) 183–191

Contents lists available at ScienceDirect

Journal of Controlled Release

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j c o n r e l

Modified pectin-based carrier for gene delivery: Cellular barriers in gene

delivery course
Tali Katav a, LinShu Liu b, Tamar Traitel a, Riki Goldbart a, Marina Wolfson c, Joseph Kost a,⁎
a
Department of Chemical Engineering, Ben-Gurion University, Beer-Sheva 84105, Israel
b
US Department of Agriculture, ARS, Eastern Regional Reaserch Center, 600 East Mermaid, Lane, Wyndmoor, PA 19038, USA
c
Department of Microbiology and Immunology, Ben-Gurion University, Beer-Sheva 84105, Israel.

A R T I C L E I N F O A B S T R A C T

Article history: The use of polysaccharides as DNA carriers has high potential for gene therapy applications. Pectin is a
Received 19 February 2008 structural plant polysaccharide heterogeneous with respect to its chemical structure. It contains branches
Accepted 2 June 2008 rich in galactose residues which serve as potential ligands for membrane receptors interaction. In order to
Available online 7 June 2008
make the anionic pectin applicable for DNA complexation, it was modified with three different amine groups
(cationic). Pectin-NH2 was prepared by modifying the galacturonic acids carboxyl groups with primary amine
Keywords:
Non-viral gene delivery
groups and further modified to generate pectin-T (TfN+H(CH3)2) and pectin-NH2-Q (QfN+(CH3)3). All three
Pectin modified pectins formed complexes with plasmid DNA as indicated by gel electrophoresis analysis. The size
Cellular barriers and morphology of pectin-NH2/DNA complexes were examined by transmission electron microscopy (TEM).
Cancer Transfection experiments were carried out with human embryonic kidney cell lines (HEK293), using plasmid
Targeted gene delivery DNA encoding for green fluorescence protein (GFP). Transfection efficiency was analyzed by flow cytometry
analysis, using FACS. Pectin-NH2-Q was the most efficient carrier. Addition of chloroquine (“lysosomotropic”
agent) to transfection medium substantially enhanced the HEK293 transfection, indicating that endocytosis
is the preferable internalization pathway and implies on the complex inability to escape the endosome.
Pectin's galactose residues contribution to transfection was examined by inhibiting pectin binding to
membrane receptors (galectins), using galactose and lactose as competitive inhibitors to this interaction.
Resulting reduction of transfection efficiency demonstrated the importance of pectin's galactose residues to
HEK293 transfection. Suggesting the modified pectin is a promising non-viral carrier for targeted gene
delivery to cancer cells with galactose-binding lectins on their surface.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
Large numbers of polymer based systems have been studied for
their physico-chemical and biochemical characteristics as well as for
their therapeutic potential. The most studied synthetic polymeric
carriers include poly-L-lysine (PLL) [1–3], polyethyleneimine (PEI)
[4,5] and polyamidoamine (PAMAM) dendrimers [5,6]. Non-viral gene
delivery systems based on natural polysaccharides may be advanta-
geous over the current available synthetic ones, due to several
characteristics, such as biodegradability, biocompatibility, low immu-
nogenicity and minimal cytotoxicity [7–9]. Furthermore, carbohy-
drate-mediated interaction with cell surface lectins play an important
role in many biological process and can be utilized to enhance the
binding step and cell uptake in a specific manner [10]. The most
studied polysaccharide carrier is chitosan. Chitosan-based delivery
systems was reported to facilitate gene transport in a cell type-
dependent manner, and proved to be safe and efficient both in vivo
⁎ Corresponding author. Tel.: +972 8 6461766; fax: +972 8 6472919.
E-mail address: kost@bgu.ac.il (J. Kost).
and in vitro [8,11,12]. Other polysaccharides, such as dextran [13] and
starch (lately studied in our lab), were chemically modified with
amine groups and their complexation and transfection activities
demonstrated encouraging results implying on their potential for
gene delivery applications.
In order to achieve efficient and specific cell uptake (gene
targeting) ligands are often attached to the carrier-DNA complex to
facilitate receptor-mediated endocytosis [14–16]. The “glycotargeting”
approach exploits the highly specific interaction between lectin
receptors and specific carbohydrates, by using carbohydrates-based
ligands, such as mannose [10], lactose [12,17] and galactose [10,18,19].
In addition to lectin receptors that are regularly involved in
endocytosis, receptors that do not directly mediate endocytosis may
also be targeted. The interaction between such receptors and the DNA
complexes will result in higher concentrations of complexes at given
cell surfaces, and therefore, increased chances for their internalization
[20].
Pectin modified with various amine groups was studied here for its
potential as a non-viral gene delivery carrier. We believe that the
galactose-rich side chains of the pectin molecule may be advantageous

0168-3659/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2008.06.002
GENE DELIVERY
184 T. Katav et al. / Journal of Controlled Release 130 (2008) 183–191
for targeted gene delivery. Pectin is a polysaccharide degraded by
colonic microflora [21], heterogeneous with respect to chemical
structure. It consists partly of homogalacturonan regions (“smooth
regions”) which are interrupted by ramified regions (“hairy regions”).
The homogalacturonan are composed of galacturonic acid residue
conferring the pectin its anionic nature. The ramified regions backbone
consists of alternating rhamnose and galacturonic acid residues.
Natural sugars such as galactose and arabinose often occur as side
chains linked to the rhamnogalacturonan portion of the pectin
backbone. In pectin from all sources, the carboxyl groups of the
galacturonic acid residues are partially methyl esterified. The degree of
esterification (DE) varies depending on the source of pectin and
isolation conditions [22–26].
Modified Citrus Pectin (MCP) has been proven to be effective in
inhibition or blocking of cancer cell aggregation, adhesion, and
metastasis [27,28]. It is believed that the short galactose-rich poly-
saccharide units confer MCP the ability to access and bind tightly to
galactose-binding lectins (galectins) on the surface of certain types of
cancer cells. To date, 14 different galectins have been characterized. Each
individual galectin is expressed in tissue-specific or developmentally
regulated fashion [29]. Therefore we believe that the binding affinity of
pectin, as well as its transfection efficiency will be cell type dependent.
The objective of this study was to develop a targeting gene carrier
system based on natural polysaccharide, pectin. Citrus pectin was
modified with different amine groups positively charged at physio-
logical pH, and complexed with plasmid DNA. The transfection
efficiency of the modified pectins was studied. The mechanism of
gene transfer and potential limiting steps to pectin-mediated
transfection, were evaluated.
2. Materials and methods
Ethanol (459844), Potassium bromide (22,186-4), quaternization
reagent 3-chloro-2-hydroxypropyltrimethylammonium chloride
(CHMAC) (348287), Sodium Chloride (S7653), α-Lactose (L-3625), D-
galactose (G-0750), Sucrose (S0389), D-glucose (G-7021), Chloroquine
diphosphat salt (C-6628), Trisodium citrate (S1804), 4-(2-hydroxy
ethyl)-1-piperazine-ethanesulfonic acid (HEPES) (H3375), Trizma
base (T6791) and EDTA (E1644) were obtained from Sigma-Aldrich,
Inc. (Israel). Galicilial acetic acid (8762) was from Gadot Biochemical
Industries Ltd. (Haifa Bay, Israel). Natural red (NR) (50040) was
purchased from Polysciences, Inc. (Warrington, PA). Ethidium bromide
(EtBr) (E40673) was purchased from TAMAR Laboratories Supplies
Ltd. (Mevaseret Zion, Israel) and Agarose (1550-027) was purchased
from GIBCO BRL, Life Technologies. Dulbecco's Modified Eagle
Medium (DMEM) (01-055-1A), RPMI 1640 medium (011001A), Fetal
calf serum (041211A), Trypsin EDTA (03-050-1B), Trypan blue (T6146),
L-glutamine (03-020-1B), penicillin-streptomycin (03-031-1B), Dul-
becco's phosphate buffered saline (PBS, 02-023-1A), 1 kb DNA lader
(G65711), loading buffer Blue/orange Dye, and 6× LB agar mixture
(G1881) were purchased from Biological Industries (Beit Haemek,
Israel). Formvar (15/95) was purchased from Ladd Research Industries
(Burlington, VT) and Uranyl acetate (22400) from Electron Microscopy
Science (Fort Washington, PA).
TAE electrophoresis buffer was prepared routinely in our lab as 50×
stock solution, by mixing 242 g Triz base, 57.1 ml Galicilial acetic acid
and 100 ml of 0.5 M EDTA pH = 8. HEPES buffered saline (HBS) solution
was prepared by dissolving 2.3 g 4-(2-hydroxy ethyl)-1-piperazine-
ethanesulfonic acid (HEPES) in 500 ml DDW containing 150 mM NaCl
to reach pH = 7.4.
2.1. Plasmid DNA
Plasmid pEGFP-C2 encoding for green fluorescence protein (GFP)
was routinely amplified in our lab in E.Coli DHα [30] and purified
using Jetstar plasmid maxi kit 10 (220010) from GIBCO BRL.
2.2. Modified pectin
Two types of modified citrus pectin (pectin-NH2 and pectin-T, TfN+
H(CH3)2) were prepared, both were previously described [31]. Briefly,
pectin macromolecule were de-esterified to reduce esterification
degree and obtain large number of carboxyl groups attached on poly
(galacturonic acid) backbone (pectin-COOH). Reaction of pectin-COOH
with ethylenediamine (1:100 mol, carboxyl/amine) using EDC as
coupling agent resulted in pectin modified with primary groups
(pectin-NH2) mainly at the pectin backbone. Pectin-NH2 reaction with
CHI3 in the presence of KHCO3 generated pectin-T (Tf[N+H(CH3)2]).
The average molecular weight of pectin-NH2 (80 kDa) was evaluated
by HPSEC (High-performance size-exclusion chromatography) with
on-line multi-angle laser light scattering as described previously [32].
The pKa value of pectin-NH2 (∼10) was evaluated by potentiometric
titration using a digital pH/milivolt meter (Model 611, Orion Research
Inc.) as described previously [33].
The primary amine group content of pectin-NH2 and pectin-T was
determined by TNBS method [34] as 600 and 100 μmol per gram sugar,
respectively. Thus, 500 μmol primary amine groups (out of 600) per
gram sugar on the pectin-NH2 were modified (with [N+H(CH3)2])
during pectin-T generation.
Modification of pectin-NH2 and unmodified citrus pectin (Sigma,
P-9311) with quaternized amine groups [-N+(CH3)3] to generate
pectin-NH2-Q and pectin-Q (Qf[N+(CH3)3]), respectively, was per-
formed according to method developed by Geresh et al. [35]. 500 mg
of pectin-NH2 was dispersed in sodium hydroxide solution (0.19 g/ml)
and the mixture was stirred continuously for 30 min at room
temperature. 9 g of quaternization reagent CHMAC (in aqueous
solution) were added, and stirring was continued for 24 h at room
temperature. One volume of reaction product was poured into four
volumes of acidified (1% HCl) ethanol, and the precipitate was washed
four times with 25 ml of 80% ethanol. The precipitate was then
dispersed in doubly distilled water and purred into a 12 kDa cutoffs
dialysis bag (D-9777 Sigma) that was placed in a vessel containing 2 L
DDW. The water was replaced 4 times with fresh double distilled
water (DDW) during 2 days of dialysis. The dialyzed product was then
dried by lyophilization (CHRIST EBTA 1-8 LOC-2M).
2.3. Chemical analysis of quaternized pectin
IR spectra were obtained with a Nicolet FT-IR spectrophotometer
(PROTÉGÉ 460). Pectin-NH2 and pectin-NH2-Q pellets were prepared
using KBr. Absorbance readings were taken at range of 400–4000 cm− 1.
(As a result of substitution, absorbance peak for C–N stretching
vibration should appear on the IR spectrum at 1476 cm− 1).
The nitrogen content of both pectin-NH2 and pectin-NH2-Q was
evaluated by Kjeldhal method [36]. Quaternized nitrogen atom
content (% weight) was calculated according to counter ions (Cl−)
content, evaluated using ion chromatograph (DIONEX DX600), and
electrochemical detector (ED50). Both pectin-NH2 and pectin-NH2-Q
solutions (0.2 mg/ml in DDW) were analyzed. Zeta potential
measurements of the modified pectins were performed using Zeta-
master (Malvern).
2.4. Modified pectin solutions
Modified pectins were dissolved in DDW or in HBS buffer at
concentration of 0.2–0.6 mg/ml and stirred overnight. Prior complexation
or transfection experiments, solutions were filtered via 0.2 μm sterile
filters (MILLIPORE-Millex) for removal of aggregates and sterilization.
Modified pectin solutions were sonicated in a microprocessor-
controlled, high-intensity 400 W ultrasonic processor (Sonics &
Materials, VCX400), equipped with a 1/2'' tapered tip. The device
was set to amplitude of 30% in a pulse mode of 0.5/0.5. Sonication
proceeded in a 20 ml glass vial, filled with 5 ml of modified pectin

T. Katav et al. / Journal of Controlled Release 130 (2008) 183–191
solutions. The vial was placed in an ice bath, and the ultrasonic tip was
dipped in the solution. During sonication, the vials remained cooled in
the ice bath and the maximum temperature measured was 40 °C for
samples treated for 60 min.
2.5. Pectin/DNA complex preparation
Complexes between modified pectin and plasmid DNA (pEGFP-C2)
were made at various pectin/DNA weight ratios (μg pectin/μg DNA).
Modified pectin solutions were added in aliquots, each account for
increasing weight ratio, to an Eppendorf tube (1.5 ml) containing DNA.
Following 3 s vortexing, the complexes were incubated at room
temperature for 1 h before use.
In the assays for complex unpacking, complexes were incubated
3 days at room temperature either with chloroquine (to final
chloroquine concentration of 100 μM) or in NaCl solutions of increasing
ionic strength (to final NaCl concentrations of: 0, 1.75, 2.25 M), or with
both chloroquine and NaCl.
2.6. Agarose gel electrophoresis
Samples containing 0.5 μg DNA either alone or complexed with
modified pectin at desired weight ratio, were mixed with loading
buffer and loaded onto 0.8% agarose gel containing ethidium bromide
(0.2 μg/ml) Samples were run in TAE running buffer, using a horizontal
gel electrophoresis apparatus (GIBCO BRL Horizon 11∙14 11068-012).
The gel was exposed to an electric field (100 V) for an hour and
visualized by UV illumination (Alpha Innotech Corporation,).
2.7. Cell culture
Human embryonic kidney cell line (HEK293) was used for
transfection experiments. Cells were grown in Dulbecco's Modified
Eagle Medium (DMEM), supplemented with 10% fetal calf serum, 1% L-
glutamine, and 1% penicillin- streptomycin. Cells were maintained in
an incubator (Tuttnauer) at 37 °C in a 5% CO2 humidified atmosphere.
2.8. Transfection experiments
24 h before transfection, 65 ⁎ 104 cells were seeded in 12 multi-well
tissue culture plates (665180, Greiner Bio-One) and grown to 60–70%
confluence. For complexes preparation, modified pectins were
dissolved in DDW or HBS (0.2–0.6 mg/ml) and added to an eppendorf
tube (1.5 ml) containing 2–4 μg DNA, to achieve desired pectin/DNA
ratio. 10 min prior transfection, the medium was aspirated from each
well, the cells were washed with PBS and covered with fresh RPMI
medium without serum. Complexes mixture was dripped gently from
the pipette into the wells, and cells were incubated for 6–24 h in an
incubator, at 37 °C in a 5% CO2 humidified atmosphere. Total volume
(medium and complex mixture) in each well was 500 μl and contained
2–4 μg DNA /well. After incubation, the transfection mixture was
removed, and the cells were overlaid with fresh medium (containing
serum) and returned to the incubator. Cells were monitored for GFP
expression using Fluorescence microscope (Leica DM IRB), and
transfection efficiency was analyzed quantitatively using Flow
Activated Cell Sorter (FACS) (Becton Dickinson FACS Calibur).
For transfection experiments involving chloroquine, calculated
amount of chloroquine diphosphate salt solution (1 mg/ml in sterile
DDW) was added to the transfection medium to final concentration of
100 μM.
For transfection experiments involving sucrose, calculated amount
of sucrose was added to the transfection medium to final concentra-
tion of: 25, 125, 200, 260 and 400 mM.
As positive control a commercial carrier were used: JetPEI®
(Polyplus Transfection Inc., New York, NY). As negative control,
naked DNA was used.
GENE DELIVERY
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2.9. Transmission electron microscopy
Transmission electron microscopy (TEM) observations were
performed using JEOL JEM-1230 Electron microscope (80 kV). Micro-
graphs were taken with a TVIPS TEMCAM-F214 camera. Pectin-NH2/
DNA complexes at calculated pectin/DNA ratio were prepared to final
DNA concentration of 10 μg/ml. 5 μl of each sample were placed onto
200 mesh copper grids, coated with formvare for 1 min, followed by
removal of excess solution. Complexes were negatively stained with
30 μl aqueous uranyl acetate solution (1%, w/w) for 20 s and excess
liquid was removed with a blotting paper.
2.10. Flow cytometry analysis
The number of cells expressing GFP was analyzed by flow
cytometry using FACS (Becton Dickinson FACS Calibur). 48 h post
transfection, cells were washed with PBS buffer and detached from the
wells with trypsin. Suspended cells were placed in FACS tubes (BD
Falcon, 352052) and centrifuged (7 min, 1000 rpm) (Jouan centrifuge,
CR412), then washed with PBS and resuspended in 0.5 ml PBS. The
fluorescence was recorded at 520 nm after an excitation at 488 nm.
10,000 cells were analyzed for each sample and results are expressed
as a number of cells expressing GFP.
2.11. Cell viability
Cytotoxicity was assessed as cell viability using Neutral Red (NR)
assay. 48 h post transfection, medium was aspirated, and cells were
washed with PBS and incubated with growth medium containing 10%
NR solution (0.21% in ethanol/DDW solution 1:100) for 2 h in a CO2
incubator, at 37 °C. After incubation, cells were washed thoroughly
with PBS, and lysed with Sorenson buffer (30.5 mM trisodium citrate,
19.4 mM HCl and 50% ethanol) for 30 min with agitation. Color
intensity was read by ELISA reader (MRX Microplate reader) at
570 nm. Results are expressed as % viability relative to untreated cells
(cells that were incubated with RPMI without serum for 6 h).
2.12. Transfection inhibition
24 h before transfection, 65 ⁎ 104 cells were seeded in 12 multi-well
tissue culture plates (665180, Greiner Bio-One) and grown to 60–70%
confluence. 60 min prior transfection, the medium was aspirated, the
cells were washed with PBS and incubated with serum-free RPMI
medium, containing 25 mM D-galactose or 50 mM lactose (competi-
tive inhibitors). Control cells were incubated with serum-free RPMI
medium without the inhibitors. 10 min before transfection, medium
was aspirated and replaced with fresh RPMI medium containing
100 μM chloroquine, with or without inhibitor. Pectin-NH2-Q/DNA
complexes (15 μg pectin/μg DNA) mixture was dripped gently into the
wells, and cells were incubated for 6 h in a CO2 incubator at 37 °C. Total
volume in each well was 500 μl, with final concentration of 100 μM
chloroquine and 0.8 μg/ml DNA. After 6 h incubation, the transfection
mixture was removed and the cells were overlaid with fresh medium
containing serum and returned to the incubator. 48 h after transfec-
tion, cells were monitored for GFP expression using fluorescence
microscope (Leica DM IRB), and transfection efficiency was analyzed
using FACS. Results are given as % cells expressing GFP relative to
control (transfection without inhibitor).
3. Results and discussion
3.1. Pectin/DNA complex formation
Complexes at various pectin/DNA ratios (pectin-NH2 and pectin-T)
were subjected to the gel retardation assay, and the migration profile
was analyzed. As the pectin/DNA ratio increases, the plasmid charge
GENE DELIVERY
186 T. Katav et al. / Journal of Controlled Release 130 (2008) 183–191

neutralizes, and its migration capability is hindered, until no free DNA

is visible (complete complexation). This can be attributed either to
complex size or to its net charge (neutral or positive). Complete
complexation of plasmid with pectin-NH2 occurred at weight ratio of
6 μg pectin/μg DNA, and at 14.2 μg pectin/μg DNA with pectin-T (for
both: 1 μg pectin /μg DNA = 0.183 N/P).
Although pectin-NH2 and pectin-T contain the same amount of
nitrogen atoms, the type of amine groups modified on the pectin
molecule is different. Complete complexation with pectin-T occurred
at higher pectin/DNA ratio than with pectin-NH2, demonstrating the
influence of amine group type on the complexation capability of the
pectin.

3.2. Transfection with pectin-NH2 and pectin-T

Transfection experiments were carried out with plasmid pEGFP-C2

encoding for green fluorescence protein (GFP) and human embryonic
kidney cell line (HEK293). Cells were monitored for GFP expression
using fluorescence microscope. Transfections with pectin-NH2/DNA
complexes were not effective. Only few cells in each well were
successfully transfected and expressed GFP. Pectin-T/DNA complexes
did not mediate transfection at any degree. For comparison,
commercially available carrier transfection was 60% for jetPEI®, and
naked DNA did not show any transfection (results not shown).
Several parameters in transfection protocol were evaluated for
their influence on transfection efficiency, including (1) concentration
of complexes in the well (2–4 μg DNA/well), (2) transfection duration
(6–24 h), (3) pectin solvent (DDW and HBS). It has been well
documented that these parameters could markedly influence trans-
fection activity and cell uptake [11]. However, in our case, combination
of the parameters mentioned above, did not enhance transfection
efficiency.
The first barrier encounter by the complexes in the course of gene
delivery is the outer cell membrane, which serves as a selective size
exclusion barrier. In order to examine whether the complex sizes may
pose an obstacle to their internalization, pectin-NH2/DNA complexes
at various ratios were subjected to Transmission Electron Microscopy
(TEM) analysis. Pectin-NH2/DNA complexes at weight ratio of 6–14 μg
pectin/μg DNA, were found to be heterogeneous with respect to their
size and morphology (Fig. 1). Major part of the population was
composed of large aggregates. In order to diminish pectin aggregation,
pectin was sonicated prior to complexation. Although sonication
markedly improved pectin solubility, aggregation was still observed in
sonicated pectin-DNA complexes (results not shown).
Aggregation may be caused either by complexes surface charge
(close to neutral), or by pectin's low solubility and tendency towards
aggregation that have been reported previously [26,37]. Complexes
aggregation may also result from bridging between particles by
external polymer loops, or by collision between electrostatic surface
patches of opposite charge on the particles. This explanation was
suggested previously for PLL and intact dendrimer mediated aggrega-
tion [5]. It seems that complexes size and complexes aggregation
prevent plasmid uptake into the cells, generating ineffective gene
delivery system.
It has been described previously that complexes bearing an
excessive positive charge, prepared at polycation/DNA ratio higher
than that necessary for complete DNA complexation, are more
effective in transfection efficiency [4]. Therefore, we used complexes
at increased pectin/DNA ratio (up to 20 μg pectin/μg DNA), however,
there was no improvement in transfection efficiency.
One explanation to these findings is the pectin molecule that is a
highly branched polysaccharide. The amine groups modified on
pectin-NH2 are located on the galacturonic acid residues on the
pectin backbone. Interactions between the pectin's amine groups and
the DNA molecule are restricted to the pectin backbone and might be
hindered by structural interferences caused by the side chains of the
Fig. 1. Transmission Electron Microscopy (TEM) of pectin-NH2/DNA complexes at
weight ratio 6 μg pectin/μg DNA. Samples were negatively stained with aqueous uranyl
acetate solution.
unmodified “hairy” region. Moreover, in comparison to other poly-
meric gene delivery systems employing macromolecules with a very
high cationic charge density [5,8,13], the amine groups content and
positive charge density on the modified pectin molecule is relatively
low. We, therefore, decided to evaluate pectin carrier with higher
content of amine groups having amine groups also on the side chains.
3.3. Pectin quaternization
In order to increase the amine groups content and their
distribution on the pectin molecule, we modified pectin-NH2 with
quaternized amine groups [-N+(CH3)3].
According to quaternization reaction [35], there are two potential
groups which are most likely to be quaternized. One is the alcohol
group, located on the galactose groups comprising the side chains at
the “hairy” region. Second is the primary amine group located at the
pectin backbone. Although the primary amine groups are more
reactive than the alcohol groups, the last ones have the advantage of
being more accessible, as they are located at the polymer branches
(Fig. 2). By quaternizing pectin-NH2, we generated third type of
modified pectin, named pectin-NH2-Q. Unmodified citrus pectin
(polyanionic with esterification degree of 26%) was also quaternized
under the same condition to generate pectin-Q.
Substitution of quaternary ammonium groups was confirmed
using FT-IR spectroscopy. As a result of substitution, the absorbance
peak for C–N stretching vibration appears on the IR spectrum at
1476 cm− 1. This peak is absent in pectin-NH2 (Fig. 3-a), and appeared
after quaternization in pectin-NH2-Q (Fig. 3-b). Quaternization of
unmodified pectin hardly changed its IR spectrum. The 1476 cm− 1
peak height, seen after pectin quaternization, was relatively small
(data not shown), indicating low degree of substitution. The more
efficient quaternization of pectin-NH2 in comparison to that of
unmodified pectin, can be related to the chemical differences between
both pectins. While unmodified pectin is polyanionic and partially
esterified, pectin-NH2 is polycationic, and unesterified.

T. Katav et al. / Journal of Controlled Release 130 (2008) 183–191
Fig. 2. Optional locations for quaternary amine group modification on pectin-NH2.
Increase of nitrogen atom content as a result of quaternization was
also confirmed by Kjeldhal method and Ion chromatography analysis.
The difference in weight percent of nitrogen atoms was 0.3% (1 μg
pectin-NH2-Q/μg DNA = 0.191 N/P). Addition of quaternized amine
groups on pectin-NH2 resulted in increased surface charge, as
confirmed by Zeta potential measurements before and after quaterni-
zation: value measured for pectin-NH2 was 20.6 ± 0.14 mV, and 34.25 ±
0.35 mV for pectin-NH2-Q.
We, therefore, conclude that quaternary ammonium groups were
modified on the pectin-NH2, generating third kind of modified pectin,
pectin-NH2-Q, with highest content of amine groups.
3.4. DNA condensation by pectin-NH2-Q and pectin-Q
Quaternized pectin (pectin-NH2-Q and pectin-Q) capability to
interact and condense DNA was evaluated using agarose gel electro-
phoresis. Complexes at various pectin/DNA ratios were loaded on the
gel, and the migration profile was analyzed.
Complete complexation was obtained at pectin-NH2-Q/DNA
weight ratio of 1.6 μg pectin/μg DNA, in comparison to 6 for pectin-
NH2 (Fig. 4). Complexes at pectin-NH2-Q weight ratio 8 μg pectin/μg
DNA were invisible in the loading well, indicating a high degree of
DNA condensation.
Fig. 3. FT-IR spectroscopy: (a) Pectin-NH2, (b) Pectin-NH2-Q.
GENE DELIVERY
187
Pectin-Q is able to complex with DNA, however, complete com-
plexation was obtained only at high pectin-Q/DNA weight ratio of 24 μg
pectin/μg DNA. This result supports the FTIR analysis, indicating that
quaternization of unmodified pectin was inefficient and, therefore,
concentration of quaternized amine groups able to interact with DNA is
relatively low.
The reduced amount of pectin-NH2-Q required for DNA complexa-
tion, in comparison to pectin-NH2, cannot only be related to the
increase in nitrogen content, but also to the improved solubility of
pectin-NH2-Q in comparison to pectin-NH2, and to the better
capability of quaternary groups to interact electrostatically with
DNA and condense it.
3.5. Transfection using pectin-NH2-Q
Transfection experiments with pectin-NH2-Q/DNA complexes
were performed with HEK293 cell lines, using plasmid pEGFP-C2.
Transfection efficiency was estimated by fluorescence microscopy and
the precise number of cells expressing GFP was determined by flow
cytometry analysis using FACS.
As evaluated by fluorescent microscope, transfection efficiency
with pectin-NH2-Q was higher than with pectin-NH2. Quantitative
analysis (48 h post transfection), using flow cytometry (FACS), has
revealed only 200 out of 10,000 HEK293 cells expressed GFP (b2%),
namely, transfection efficiency was still very low. GFP expression was
monitored for 5 days after transfection and no increase in transfection
efficiency was observed during this period.
Although optimization of the transfection protocol resulted in
improved transfection efficiency, the actual percentage of cells expres-
sing GFP was still relatively low, therefore, we evaluate several cellular
barriers that may be limiting pectin-NH2-Q mediated transfection.
4. Cellular barriers to pectin-NH2-Q transfection
4.1. Endosomal escape
Numerous reports have described the design and synthesis of
polymers suggesting the escape from endocytic vesicles as the limiting
step in the transfection process [8,12,38,39]. Several of these suggestions
GENE DELIVERY
188 T. Katav et al. / Journal of Controlled Release 130 (2008) 183–191

Fig. 4. Agarose gel electrophoresis of Pectin-NH2-Q/DNA complexes at increased weight

ratios.

were based on the “proton sponge” hypothesis, which asserts that certain
polymers such as PEI, containing large number of unprotonated basic
groups (typically amines) may prevent acidification of endocytic vesicles.
Endosome buffering leads to the formation of suboptimal environment
required for lysosomal nuclease activation, thus preventing DNA
degradation. Furthermore, an increase in the osmotic pressure within
the endocytic vesicle may result in vesicle swelling and induce polyplexes
Fig. 5. Microscopic examination of HEK293 cells, 48 h post transfection with pectin-
release to the cytosol [2,4,8,38,40].
NH2-Q complexes (15 μg pectin/μg DNA): (a) without chloroquine, and (b) in the
Since both primary and quaternized amine groups of the pectin-NH2-Q presence of 100 μM chloroquine. Cells were visualized by fluorescent (left) and visible
are protonated at physiological pH, pectin-NH2-Q is unlikely to have any light (right). The right image is a composition of both, fluorescent and light images.
buffering capacity at the acidic pH of the endocytic vesicles. Unless pectin-
NH2-Q /DNA complexes can escape the endosome by some other on its ability to raise the osmotic pressure inside the cytoplasmic
mechanism than the “proton sponge mechanism”, they will most likely vesicles and induce DNA complexes release to the cytosol [43].
be degraded in the lysosome before the DNA could reach the nucleus. Sucrose presence in transfection medium (25, 125, 200, 260 and
In order to confirm our hypothesis we use the “lysosomotropic agent” 400 mM) did not enhance transfection. Furthermore, as the sucrose
chloroquine, which has been reported to be effective in several polymeric concentration increased, the toxicity was elevated as well. Apparently,
gene delivery systems [1,2,38,41–43]. Chloroquine is known to interfere although both chloroquine and sucrose are “lysosomotropic” agents,
with the endocytosis process, in particular, raising the luminal pH of however, only chloroquine succeeded to increase transfection effi-
endocytic vesicles and reducing ligand delivery to lysosomes, thereby ciency. These results may be related to the different mechanism of
protecting DNA molecules from nuclease degradation by lysosomal action of the lysosomotropic agents. It has been suggested that
enzymes [1,2,43]. It has been suggested that, when the concentration of chloroquine may also be involved in complexes “unpacking”, which is
chloroquine in vesicles becomes sufficiently high, vesicles are induced to another known limiting step to transfection [1].
swell, resulting in plasmid released to the cytosol [1].
For transfection experiments involving chloroquine, calculated
amount of chloroquine was added to the transfection medium to
desired final concentration of 100 μM. Other transfection conditions
were according to optimization results obtained previously. HEK293
transfection efficiency was significantly improved in the presence of
chloroquine (Fig. 5).
As can be seen in Fig. 6, chloroquine increases transfection by
twenty fold on average. Complexes at weight ratio of 15 μg pectin/μg
DNA resulted in the highest transfection efficiency (Fig. 7). Although
chloroquine markedly enhanced transfection effectiveness efficiency
in vitro, its use in vivo is limited by its cytotoxic effect [2,43]. Cell
viability studies (Fig. 8) demonstrated pectin-NH2-Q only slightly
decreases viability (5%), while transfection with pectin-NH2-Q/DNA
complexes reduces cell viability by 12%. It is important to notice that
pectin-NH2-Q, which is a positively charged polysaccharide, is not
toxic to the cells. When chloroquine was added to the medium, either
alone or in combination with pectin-NH2-Q and pectin-NH2-Q/DNA
complexes (transfection with chloroquine), significant reduction of
viability was observed (about 50%).
Sucrose, another “lysosomotropic” agent, was found to be most
ideal in terms of increased gene expression, with no significant Fig. 6. Chloroquine effect on transfection efficiency: HEK293 cells 48 h post transfection
with pectin-NH2-Q complexes (15 μg pectin/μg DNA) with or without 100 μM
cytotoxic effects on cultured fibroblasts, when compared to chlor- chloroquine. Transfection efficiency was determined by GFP expression. Each experi-
oquine [43]. Sucrose was reported to increase transfection when mixed ment was performed several times (n) in duplicates; Data presented as mean (n ≥ 4) ±
with lipofectamine/DNA complexes. Its mechanism of action is based S.E.M (sample size = 10,000 cells, SEM lower than 3%).

T. Katav et al. / Journal of Controlled Release 130 (2008) 183–191
Fig. 7. Pectin-NH2-Q/DNA ratio effect on transfection efficiency : HEK293 cells were
transfected with pectin-NH2-Q complexes (5–21 μg pectin/μg DNA) in the presence of
100 μM chloroquine and analyzed 48 h later by FACS. Each experiment was performed
several times (n) in duplicates. Data presented as mean (n ≥ 4) ± S.E.M (sample
size = 10,000 cells, SEM lower than 3%).
4.2. Complex unpacking
Complexation of plasmid DNA with cationic lipids or polymers
provide protection against enzymatic degradation, however, it is
important to control the dissociation of plasmid DNA from the cationic
carrier, in order to enhance nuclear delivery and transgene expression.
If the dissociation occurs too rapidly, most of the plasmid DNA will be
degraded in the cytosol, whereas if the dissociation occurs too slowly
or not at all, the plasmid will be inaccessible to transcription factors in
the nucleus [14,39,42]. The stability of the complexes is dominated by
the strength of electrostatic interactions between the DNA and the
polycationic carrier [5]. In order to evaluate the magnitude of these
interactions, we used non-cellular system, in which pectin-NH2-Q/
DNA complexes were subjected to either increasing ionic strength
upon adding NaCl or chloroquine. Complexes integrity was estimated
using electrophoresis in EtBr containing gels. Intercalation of EtBr
Fig. 8. The effect of pectin-NH2-Q, pectin-NH2-Q/DNA complexes and chloroquine on
HEK293 cells viability. Cells incubated in RPMI medium were used as control
(“Untreated”). Cells were exposed to 60 μg pectin-NH2-Q either alone or complexed
with 4 μg DNA, with or without 100 μM chloroquine. Each experiment was performed
several times (n) and included duplicates. Data presented as mean (n ≥ 4) ± STDEV.
GENE DELIVERY
189
between DNA strands is limited when the DNA is highly condensed by
the polycation leading to a decrease in band fluorescence.
The intensity of fluorescence band in the loading wells increased
with ionic strength, indicating that the binding affinity of the cationic
pectin for the DNA was reduced. However, complexes were not
completely dissociated even in the presence of 2.25 M NaCl (no free
DNA was visible), demonstrating the interaction strength between the
modified pectin and the DNA. Such high stability of complex and low
transfection efficiency, has been reported before for DNA complexes
with other polysaccharides, such as chitosan [44] which was extremely
stable when subjected to 3 M NaCl. Erbacher et al. [1], have
demonstrated that the strong effect of chloroquine on gene transfection
efficiency is not only related to the neutralization of the vesicles but also
to complex dissociation. They demonstrated that chloroquine can
destabilize polymer/DNA complexes and enhance “unpacking” of the
polyplexes [1]. When pectin-NH2-Q/DNA complexes were subjected to
100 μM chloroquine (same concentration as in the transfection
medium), they remained highly condensed, indicating that they were
not dissociated in the medium during transfection. This result is similar
to the results reported by Erbacher et al. [1]. However, chloroquine tends
to accumulate within acidic vesicles and reach higher concentration
values (∼50 mM). Erbacher et al. showed that in the presence of 20 mM
chloroquine, DNA complexes were nearly fully dissociated. According to
our results, it seems that pectin-NH2-Q/DNA complex stability may pose
a rate limiting step to transfection, and, as Erbacher suggested, it is
possible that the mechanism of chloroquine action involves pectin-NH2-
Q/DNA complex unpacking.
5. Contribution of pectin galactose residues to transfection
Galactose-rich side chains on pectin molecule were reported to
mediate specific carbohydrate-receptor interaction with lectins
(galectins) [27,28]. Transfection efficiency of pectin-NH2-Q may be
related to its galactose-rich side chains and their interaction with
cellular galectins [29,45]. Even though galectins may not be directly
involved in endocytosis, they can localize higher concentrations of
pectin complexes at cell surface [20]. This will raise the chances for
complexes internalization and promote transfection.
In order to investigate the contribution of pectin's galactose residues
to transfection, we used galactose-containing carbohydrates as compe-
titive inhibitors to pectin–galectin interaction. Both lactose and D-
galactose have been reported to compete and inhibit galectin-mediated
interaction and interfere with cell recognition and adhesion processes
[27,46,47]. In order to obtain cell surface receptors blocking (saturation),
HEK293 cells were incubated with RPMI medium containing lactose
Fig. 9. Competitive inhibition of pectin–galectin interaction: (FACS analysis). Transfec-
tion efficiency in the presence of lactose and galactose is expressed as % of transfection
efficiency performed without any inhibitor (expressed as 100%). The transfection
medium contains D-glucose, therefore it represents a negative control (“No inhibitor”).
Each experiment was performed twice in duplicates. Data presented as a mean of the
two experiments with repetition ± STDEV.
GENE DELIVERY
190 T. Katav et al. / Journal of Controlled Release 130 (2008) 183–191
(50 mM) or D-galactose (25 mM), 1 h prior transfection. The inhibitor
was also present during transfection.
Both lactose and D-galactose have decreased the level of transfec-
tion (Fig. 9), suggesting that pectin–galectin interaction may con-
tribute to complex internalization.
6. Conclusions
Pectin modified with various amine groups was studied here for its
potential as a non-viral gene delivery carrier. Based on the attractive
characteristics of the pectin molecule [27,28] and on other poly-
saccharide-based gene delivery systems [8,12,13,20], we assumed that
cationic pectin will be able to interact with DNA to form compact
transportable unit that will also be biocompatible and biodegradable.
The complexation and transfection abilities of different modified
pectins were evaluated. All modified pectins were able to interact with
plasmid DNA to form complexes, however complexation and
transfection efficiency was influenced by the amine group type,
modified on each of the pectins. Pectin-NH2-Q was able to interact
strongly with DNA, with complete complexation at the lowest weight
ratio (1.6 μg pectin/μg DNA). Pectin-NH2-Q resulted in higher
transfection efficiency than the other modified pectins. We evaluated
cellular barriers that may be limiting transfection, including endoso-
mal escape and complex unpacking. Chloroquine markedly enhanced
transfection efficiency, indicating that pectin-NH2-Q complexes are
internalized via endocytosis, but unable to avoid degradation in the
acidic lysosome. Despite chloroquine effectiveness in vitro, its use in
vivo is limited by its cytotoxic effect [2,43]. Other methods, such as
incorporation of endosomal disrupting peptides (GALA and KALA),
may be used to enhance transfection by facilitating DNA release from
the endocytic compartments with less cytotoxicity [14,48].
The pectin-NH2-Q complexes exhibited extremely high stability
when challenged with high salt concentrations (up to 2.25 M),
suggesting that complex dissociation may be another limiting step to
transfection. It has been reported that polymer molecular weight
affects its interaction with DNA, and that the stability of the complex
increases with polymer molecular weight [49,50]. Lowering the
molecular weight of pectin may reduce complex stability and increase
transfection efficiency.
In a competitive inhibition assay made with galactose and lactose,
transfection was inhibited, emphasizing galactose residues contribu-
tion to transfection. The natural galactose residues of the pectin
molecule may be exploited for gene targeting. The lectin receptors
expressed on each cell type will have different affinity towards pectin
complexes, resulting in cell type specific transfection.
In conclusion, modified pectin seems to be a promising carrier,
attractive for targeted gene delivery applications. We believe that
additional structural modifications of the carrier could significantly
farther improve its transfection efficiency.
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