GENE DELIVERY

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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 b c

Department of Chemical Engineering, Ben-Gurion University, Beer-Sheva 84105, Israel US Department of Agriculture, ARS, Eastern Regional Reaserch Center, 600 East Mermaid, Lane, Wyndmoor, PA 19038, USA Department of Microbiology and Immunology, Ben-Gurion University, Beer-Sheva 84105, Israel.

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I N F O

Article history: Received 19 February 2008 Accepted 2 June 2008 Available online 7 June 2008 Keywords: Non-viral gene delivery Pectin Cellular barriers Cancer Targeted gene delivery

A B S T R A C T The use of polysaccharides as DNA carriers has high potential for gene therapy applications. Pectin is a structural plant polysaccharide heterogeneous with respect to its chemical structure. It contains branches rich in galactose residues which serve as potential ligands for membrane receptors interaction. In order to 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 groups and further modified to generate pectin-T (TfN+H(CH3)2) and pectin-NH2-Q (QfN+(CH3)3). All three modified pectins formed complexes with plasmid DNA as indicated by gel electrophoresis analysis. The size and morphology of pectin-NH2/DNA complexes were examined by transmission electron microscopy (TEM). Transfection experiments were carried out with human embryonic kidney cell lines (HEK293), using plasmid 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 advantageous over the current available synthetic ones, due to several characteristics, such as biodegradability, biocompatibility, low immunogenicity and minimal cytotoxicity [7–9]. Furthermore, carbohydrate-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 typedependent manner, and proved to be safe and efficient both in vivo ⁎ Corresponding author. Tel.: +972 8 6461766; fax: +972 8 6472919. E-mail address: [email protected] (J. Kost). 0168-3659/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2008.06.002

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

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Journal of Controlled Release 130 (2008) 183–191

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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 polysaccharide 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 physiological 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), Dgalactose (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), Dulbecco'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-piperazineethanesulfonic 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 performed 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 Zetamaster (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 microprocessorcontrolled, 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

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,).

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2.9. Transmission electron microscopy Transmission electron microscopy (TEM) observations were performed using JEOL JEM-1230 Electron microscope (80 kV). Micrographs 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

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% Lglutamine, and 1% penicillin- streptomycin. Cells were maintained in an incubator (Tuttnauer) at 37 °C in a 5% CO2 humidified atmosphere.

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).

2.8. Transfection experiments

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. 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 concentration 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.

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 (competitive 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 transfection, 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).

2.7. Cell culture

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

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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 transfection 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 aggregation [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 polymeric 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.

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Pectin-Q is able to complex with DNA, however, complete complexation 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 complexation, 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. 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 quaternization: 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 electrophoresis. 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 pectinNH2 (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.

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 expressing 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

Fig. 3. FT-IR spectroscopy: (a) Pectin-NH2, (b) Pectin-NH2-Q.

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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 release to the cytosol [2,4,8,38,40]. Since both primary and quaternized amine groups of the pectin-NH2-Q are protonated at physiological pH, pectin-NH2-Q is unlikely to have any buffering capacity at the acidic pH of the endocytic vesicles. Unless pectinNH2-Q /DNA complexes can escape the endosome by some other mechanism than the “proton sponge mechanism”, they will most likely be degraded in the lysosome before the DNA could reach the nucleus. In order to confirm our hypothesis we use the “lysosomotropic agent” chloroquine, which has been reported to be effective in several polymeric gene delivery systems [1,2,38,41–43]. Chloroquine is known to interfere with the endocytosis process, in particular, raising the luminal pH of endocytic vesicles and reducing ligand delivery to lysosomes, thereby protecting DNA molecules from nuclease degradation by lysosomal enzymes [1,2,43]. It has been suggested that, when the concentration of chloroquine in vesicles becomes sufficiently high, vesicles are induced to 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 cytotoxic effects on cultured fibroblasts, when compared to chloroquine [43]. Sucrose was reported to increase transfection when mixed with lipofectamine/DNA complexes. Its mechanism of action is based

Fig. 5. Microscopic examination of HEK293 cells, 48 h post transfection with pectinNH2-Q complexes (15 μg pectin/μg DNA): (a) without chloroquine, and (b) in the presence of 100 μM chloroquine. Cells were visualized by fluorescent (left) and visible light (right). The right image is a composition of both, fluorescent and light images.

on its ability to raise the osmotic pressure inside the cytoplasmic vesicles and induce DNA complexes release to the cytosol [43]. Sucrose presence in transfection medium (25, 125, 200, 260 and 400 mM) did not enhance transfection. Furthermore, as the sucrose concentration increased, the toxicity was elevated as well. Apparently, although both chloroquine and sucrose are “lysosomotropic” agents, however, only chloroquine succeeded to increase transfection efficiency. These results may be related to the different mechanism of action of the lysosomotropic agents. It has been suggested that chloroquine may also be involved in complexes “unpacking”, which is another known limiting step to transfection [1].

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 chloroquine. Transfection efficiency was determined by GFP expression. 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%).

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.

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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-NH2Q/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 competitive inhibitors to pectin–galectin interaction. Both lactose and Dgalactose 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). Transfection 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.

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(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 transfection (Fig. 9), suggesting that pectin–galectin interaction may contribute 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 polysaccharide-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 endosomal 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 contribution 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. References [1] P. Erbacher, A.C. Roche, M. Monsigny, P. Midoux, Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes, Exp. Cell Res. 225 (1996) 186–194. [2] M.L. Forrest, D.W. Pack, On the kinetics of polyplex endocytic trafficking: implications for gene delivery vector design, Mol. Ther. 6 (2002) 57–66. [3] E. Wagner, Effects of membrane-active agents in gene delivery, J. Control. Release 53 (1998) 155–158. [4] C. Zhang, P. Yadava, J. Hughes, Polyethylenimine strategies for plasmid delivery to brain-derived cells, Methods 33 (2004) 144–150. [5] M.X. Tang, F.C. Szoka, The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes, Gene Ther. 4 (1997) 823–832. [6] J. Dennig, E. Duncan, Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers, J. Biotechnol. 90 (2002) 339–347. [7] T. Azzam, H. Eliyahu, A. Makovitzki, M. Linial, A.J. Domb, Hydrophobized dextranspermine conjugate as potential vector for in vitro gene transfection, J. Control. Release 96 (2004) 309–323.

[8] M. Koping-Hoggard, I. Tubulekas, H. Guan, K. Edwards, M. Nilsson, K.M. Varum, P. Artursson, Chitosan as a nonviral gene delivery system. Structure–property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo, Gene Ther. 8 (2001) 1108–1121. [9] G. Borchard, Chitosans for gene delivery, Adv. Drug Deliv. Rev. 52 (2001) 145. [10] M. Monsigny, P. Midoux, R. Mayer, A.C. Roche, Glycotargeting: influence of the sugar moiety on both the uptake and the intracellular trafficking of nucleic acid carried by glycosylated polymers, Biosci. Rep. 19 (1999) 125–132. [11] T. Sato, T. Ishii, Y. Okahata, In vitro gene delivery mediated by chitosan. effect of pH, serum, and molecular mass of chitosan on the transfection efficiency, Biomaterials 22 (2001) 2075–2080. [12] P. Erbacher, S. Zou, T. Bettinger, A.M. Steffan, J.S. Remy, Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability, Pharm. Res. 15 (1998) 1332–1339. [13] T. Azzam, H. Eliyahu, L. Shapira, M. Linial, Y. Barenholz, A.J. Domb, Polysaccharideoligoamine based conjugates for gene delivery, J. Med. Chem. 45 (2002) 1817–1824. [14] M.D. Brown, A.G. Schatzlein, I.F. Uchegbu, Gene delivery with synthetic (non viral) carriers, Int. J. Pharm. 229 (2001) 1–21. [15] S.M. Moghimi, A.R. Rajabi-Siahboomi, Recent advances in cellular, sub-cellular and molecular targeting, Adv. Drug Deliv. Rev. 41 (2000) 129–133. [16] F.D. Ledley, Nonviral gene therapy: the promise of genes as pharmaceutical products, Hum. Gene Ther. 6 (1995) 1129–1144. [17] D.T. Klink, S. Chao, M.C. Glick, T.F. Scanlin, Nuclear translocation of lactosylated poly-L-lysine/cDNA complex in cystic fibrosis airway epithelial cells, Mol. Ther. 3 (2001) 831–841. [18] T. Ren, G. Zhang, D. Liu, Synthesis of galactosyl compounds for targeted gene delivery, Bioorg. Med. Chem. 9 (2001) 2969–2978. [19] J. Han, Y. Il Yeom, Specific gene transfer mediated by galactosylated poly-L-lysine into hepatoma cells, Int. J. Pharm. 202 (2000) 151–160. [20] B.G. Davis, M.A. Robinson, Drug delivery systems based on sugar-macromolecule conjugates, Curr. Opin. Drug Discov. Dev. 5 (2002) 279–288. [21] L. Liu, M.L. Fishman, J. Kost, K.B. Hicks, Pectin-based systems for colon-specific drug delivery via oral route, Biomaterials 24 (2003) 3333–3343. [22] E. Bonnin, E. Dolo, A. Le Goff, J.F. Thibault, Characterisation of pectin subunits released by an optimised combination of enzymes, Carbohydr. Res. 337 (2002) 1687–1696. [23] B.L. Ridley, M.A. O'Neill, D. Mohnen, Pectins: structure, biosynthesis, and oligogalacturonide-related signaling, Phytochemistry 57 (2001) 929–967. [24] J.M. Ros, H.A. Schols, G.J. Voragen, Extraction, characterisation, and enzymatic degradation of lemon peel pectins, Carbohydr. Res. 282 (1996) 271–284. [25] H.A. Schols, E. Vierhuis, E.J. Bakx, A.G. Voragen, Different populations of pectic hairy regions occur in apple cell walls, Carbohydr. Res. 275 (1995) 343–360. [26] M.L. Fishman, H.K. Chau, P. Hoagland, K. Ayyad, Characterization of pectin, flashextracted from orange albedo by microwave heating, under pressure, Carbohydr. Res. 323 (2000) 126–138. [27] D. Platt, A. Raz, Modulation of the lung colonization of B16-F1 melanoma cells by citrus pectin, J. Natl. Cancer Inst. 84 (1992) 438–442. [28] P. Nangia-Makker, V. Hogan, Y. Honjo, S. Baccarini, L. Tait, R. Bresalier, A. Raz, Inhibition of human cancer cell growth and metastasis in nude mice by oral intake of modified citrus pectin, J. Natl. Cancer Inst. 94 (2002) 1854–1862. [29] H. Lahm, S. Andre, A. Hoeflich, J.R. Fischer, B. Sordat, H. Kaltner, E. Wolf, H.J. Gabius, Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures, J. Cancer Res. Clin. Oncol. 127 (2001) 375–386. [30] J. Sambrook, D.W. Russell, Molecular cloning, Vol. 1, Cold Spring Harbor Laboratory Press, New York, 2001 1.116 pp. [31] L.S. Liu, Y. Ito, Y. Imanishi, Synthesis and antithrombogenicity of heparinized polyurethanes with intervening spacer chains of various kinds, Biomaterials 12 (1991) 390–396. [32] L. Liu, Y.J. Won, P.H. Cooke, D.R. Coffin, M.L. Fishman, K.B. Hicks, P.X. Ma, Pectin/ poly(lactide-co-glycolide) composite matrices for biomedical applications, Biomaterials 25 (2004) 3201–3210. [33] L. Liu, M.L. Fishman, K.B. Hicks, M. Kende, Interaction of various pectin formulations with porcine colonic tissues, Biomaterials 26 (2005) 5907–5916. [34] L.S. Liu, C.K. Ng, A.Y. Thompson, J.W. Poser, R.C. Spiro, Hyaluronate-heparin conjugate gels for the delivery of basic fibroblast growth factor (FGF-2), J. Biomed. Mater. Res. 62 (2002) 128–135. [35] S. Geresh, R.P. Dawadi, S.M. Arad, Chemical modifications of biopolymers: quaternization of the extracellular polysaccharide of the red microalga Porphyridium sp. Carbohydr. Polym. 63 (2000) 75–80. [36] A.I. Vogel, A textbook of quantitative inorganic analysis, Longman, London, 1961, pp. 256–257. [37] A.N. Round, N.M. Rigby, A.J. MacDougall, S.G. Ring, V.J. Morris, Investigating the nature of branching in pectin by atomic force microscopy and carbohydrate analysis, Carbohydr. Res. 331 (2001) 337–342. [38] P.C. Zhang, J. Wang, K.W. Leong, H.Q. Mao, Ternary complexes comprising polyphosphoramidate gene carriers with different types of charge groups improve transfection efficiency, Biomacromolecules 6 (2005) 54–60. [39] H. Kamiya, H. Akita, H. Harashima, Pharmacokinetic and pharmacodynamic considerations in gene therapy, Drug Discov. Today 8 (2003) 990–996. [40] A.R. Klemm, D. Young, J.B. Lloyd, Effects of polyethyeneimine on endocytosis and lysosome stability, Biochem. Pharmacol. 56 (1998) 41–46. [41] T.M. Reineke, M.E. Davis, Structural effects of carbohydrate-containing polycations on gene delivery. 2. Charge center type, Bioconjug. Chem. 14 (2003) 255–261. [42] J. Wen, H.Q. Mao, W. Li, K.Y. Lin, K.W. Leong, Biodegradable polyphosphoester micelles for gene delivery, J. Pharm. Sci. 93 (2004) 2142–2157.

[43] K. Ciftci, R.J. Levy, Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts, Int. J. Pharm. 218 (2001) 81–92. [44] F.C. MacLaughlin, R.J. Mumper, J. Wang, J.M. Tagliaferri, I. Gill, M. Hinchcliffe, A.P. Rolland, Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery, J. Control. Release 56 (1998) 259–272. [45] J.L. Wang, R.M. Gray, K.C. Haudek, R.J. Patterson, Nucleocytoplasmic lectins, Biochim. Biophys. Acta 1673 (2004) 75–93. [46] P. Nangia-Makker, Y. Honjo, R. Sarvis, S. Akahani, V. Hogan, K.J. Pienta, A. Raz, Galectin-3 induces endothelial cell morphogenesis and angiogenesis, Am. J. Pathol. 156 (2000) 899–909. [47] D. Avichezer, R. Arnon, Differential reactivities of the Arachis hypogaea (peanut) and Vicia villosa B4 lectins with human ovarian carcinoma cells, grown either in vitro or in vivo xenograft model, FEBS Lett. 395 (1996) 103–108.

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[48] S. Simoes, V. Slepushkin, P. Pires, R. Gaspar, M.P. de Lima, N. Duzgunes, Mechanisms of gene transfer mediated by lipoplexes associated with targeting ligands or pH-sensitive peptides, Gene Ther. 6 (1999) 1798–1807. [49] M. Koping-Hoggard, Y.S. Mel'nikova, K.M. Varum, B. Lindman, P. Artursson, Relationship between the physical shape and the efficiency of oligomeric chitosan as a gene delivery system in vitro and in vivo, J. Gene Med. 5 (2003) 130–141. [50] D.V. Schaffer, N.A. Fidelman, N. Dan, D.A. Lauffenburger, Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery, Biotechnol. Bioeng. 67 (2000) 598–606.

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