Tailoring of Chitosans for Gene Delivery: Novel Self-Branched

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Biomacromolecules 2008, 9, 3268–3276

Tailoring of Chitosans for Gene Delivery: Novel Self-Branched Glycosylated Chitosan Oligomers with Improved Functional Properties Sabina P. Strand,*,†,‡ Mohamed M. Issa,‡ Bjørn E. Christensen,† Kjell M. Vårum,† and Per Artursson‡ Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway, and Department of Pharmacy, Uppsala University, Box 580, 751 23 Uppsala, Sweden Received July 24, 2008; Revised Manuscript Received August 26, 2008

Chitosan is a promising biomaterial with an attractive safety profile; however, its application potential for gene delivery is hampered by poor compatibility at physiological pH values. Here we have tailored the molecular architecture of chitosan to improve the functional properties and gene transfer efficacy of chitosan oligomers and have developed self-branched glycosylated chitosan oligomer (SB-TCO) substituted with a trisaccharide containing N-acetylglucosamine, AAM. SB-TCO was prepared by controlled depolymerization of chitosan, followed by simultaneous branching and AAM substitution. The product was fully soluble at physiological pH and complexed plasmid DNA into polyplexes of high colloidal and physical stability. SB-TCO displayed high transfection efficacy in HEK293 cells, reaching transfection efficiencies of up to 70%, and large amounts of transgene were produced. Gene transfer efficacy was confirmed in HepG2 cells, where gene expression levels mediated by SB-TCO were up to 10 and 4 times higher than those obtained with unsubstituted and substituted linear oligomers, respectively. The rapid onset of transgene expression in both cell lines indicates efficient DNA release and transcription from SB-TCO polyplexes. In comparison with 22 kDa linear PEI-based transfection reagent used as the control, SBTCO possessed higher gene transfer efficacy, significantly lower cytotoxicity, and improved serum compatibility.

Introduction Gene delivery into mammalian cells has become an indispensable research tool in molecular and cell biology. Despite widespread use and numerous delivery systems available, transfection is still a matter of compromise between acceptable toxicity and efficacy. Compared with viral vectors, nonviral carriers are considerably safer and easy to produce, and they possess large gene-carrying capacity and flexibility of design. However, most of these transfection reagents exhibit significant cytotoxicity, which is often correlated with transfection efficacy.1-6 Because lack of toxicity is a major demand in the design of new gene delivery systems, the development of nonviral vectors has been increasingly focused on biocompatible systems and natural polymers.7 These include polysaccharides such as schizophyllan,8 glycopolymers,9,10 or degradable synthetic polymers.2,11-14 Among the biopolymers, chitosans, a family of cationic and linear polysaccharides derived from chitin, have received increasing attention in biomedical research over the past decade15-17 and show an attractive safety profile as well as a range of possibilities for further modifications.18-21 The intrinsic properties of a particular chitosan sample strongly depend on structural variables such as the fraction of acetylated units (FA) and the degree of polymerization (DP), and accordingly, both the FA and the DP have been shown to have a large impact on the physicochemical and biological activities of the polymer.20,22,23 Unfortunately, the application of chitosan in biological systems is generally hampered by its poor compatibility at physiological * Corresponding author. E-mail: [email protected]. † Norwegian University of Science and Technology. ‡ Uppsala University.

pH. Compared with other amine-containing polyelectrolytes such as polyethylenimine (PEI) and poly-L-lysine (PLL), the primary amino group of the GlcN units possesses a relatively low pKa value of 6.5.24 Accordingly, the charge density of chitosan at pH 7.4 is very low, and generic chitosans are insoluble. To overcome this limitation, different derivatives of chitosan have been prepared, including trimethylated25-27 or pegylated chitosans.25,28,29 However, substantial modification of the polycation structure has been shown to interfere with both DNA condensation30 and cell uptake.31 Quaternization of chitosan also lead to increased cytotoxicity.25,26 The broad variety of available chitosans and their structuredependent sensitivity to pH and ionic conditions may be a reason for the highly variable efficacy of chitosan-based gene delivery systems reported in the literature. Although most chitosans are able to compact DNA into nanosized polyplexes, the stability and properties of the formed polyplexes strongly depend on chitosan structural variables.32-36 Whereas conventional highmolecular-weight chitosans have been found to possess low gene delivery efficacy both in vitro and in vivo,22,25,37 tailoring of structural variables (FA, DP, polydispersity) and optimization of transfection protocols have been shown to yield highly efficient gene delivery systems in different application areas in vitro and in vivo.38-40 It has been suggested that a key to successful transfection with chitosan is to achieve a subtle balance between DNA protection and intracellular DNA release.36,39 Consequently, all variables that affect the stability of polyplexes such as chitosan molecular parameters, composition of DNA/chitosan complexes, details of the transfection protocol, and so on were found to influence gene transfer efficacy.35,38-41

10.1021/bm800832u CCC: $40.75  2008 American Chemical Society Published on Web 10/04/2008

Tailoring of Chitosans for Gene Delivery

Recently, chitosan oligomers substituted with a GlcNAccontaining trisaccharide denoted AAM were reported to target membrane-bound lectins in airway epithelial cells, improving gene delivery efficacy compared with unsubstituted oligomers.38 Furthermore, glycosylation of chitosan was also shown to improve the colloidal stability of the formulation.38,42 However, preliminary structure-function studies with oligosaccharidesubstituted oligomers revealed a large variation in gene transfer efficacy and indicated a need for further molecular tailoring of the chitosan structure. To address this issue, we have synthesized a new generation of chitosan-based transfection reagents, self-branched glycosylated (trisaccharide-substituted) chitosan oligomers, with improved functional properties. To demonstrate the potential of these novel DNA carriers, we investigated the physicochemical properties of formulated DNA nanoparticles and compared them with those formed by substituted and unsubstituted linear oligomers. Next, we evaluated the gene delivery efficacy in HEK293 and HepG2 cells, using linear and branched PEI as controls. Here we report that by self branching of the glycosylated chitosans, we are able to eliminate several drawbacks that are inherent to chitosan and to produce a highly efficient gene delivery system.

Materials and Methods Plasmid DNA. Reporter plasmids (gWiz Luc and gWiz GFP) containing a cytomegalovirus promoter (CMV) and firefly luciferase (Luc) or green fluorescent protein (GFP) were purchased from Aldevron (Fargo, ND). Chitosans. Linear chitosan oligomers with number-average degrees of polymerization (DPn) in the range of 18-100 were prepared by nitrous acid depolymerization of fully de-N-acetylated chitosan (FA < 0.002), as described earlier.43 To prepare the glycosylated oligomers, selected depolymerized chitosan (DPn34) was reduced by NaBH4 and was reductively N-alkylated with the trimer 2-acetamido-2-deoxy-Dglucopyranosyl-β-(1-4)-2-acetamido-2-deoxy-D-glucopyranosyl-β-(1-4)2,5-anhydro-D-mannofuranose (AAM), as previously described.44 In the case of the self-branched glycosylated oligomers, the reduction step after HONO degradation was omitted, and the depolymerized chitosan (DPn34) was simultaneously self branched and substituted with AAM through 2,5-anhydro-D-mannose at the reducing end, as shown in Scheme 1. All samples were characterized by 1H NMR to determine the degree of substitution. The average degrees of polymerization and chain length distributions were analyzed by size-exclusion chromatography (SEC) with a refractive index (RI, Dawn Optilab 903, Wyatt Technology) and a multiangle laser light scattering detector (MALLS, Dawn DSP, Wyatt Technology).45 All samples were dissolved in MQ water (5-7 mg/mL) and were filtered through a 0.22 µm syringe filter (Millipore). The column used was TSK 3000 PWXL, and the sample was eluted with 0.2 M ammonium acetate (pH 4.5) at a flow rate of 0.5 mL/min. For simplicity, the three different classes of chitosan oligomers are denoted as linear, trisaccharide-substituted chitosan oligomers (TCO), and self-branched trisaccharide-substituted chitosan oligomers (SBTCO). The characteristics of the chitosans used in the study are given in Table 1. Other Transfection Reagents. PEI (25 kDa) was purchased from Sigma-Aldrich. Exgen, a commercial transfection reagent that is based on linear PEI (22 kDa), was purchased from Fermentas (Burlington, Canada). Both reagents were used according to manufacturer specifications. Solubility of Chitosans. Aliquots (50 µL) of a 2 mg/mL solution of chitosans in MQ water were transferred to a 96-well plate and were diluted to 100 µL by double-strength Hanks’ balanced salt solution (HBSS, Gibco Invitrogen) that was supplemented with 20 mM HEPES (Sigma-Aldrich) with a pH in the range of 6.5- 8.0. After the solution

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Scheme 1. Preparation of Self-Branched Chitosans by Nitrous Acid Degradation and Reductive Alkylation

Table 1. Characterization of Chitosan Samples Useda sample

d.s.

Mn

Mw

PDI

linear TCO SB-TCO

7.3 7.3

7400 8600 13 700

9700 11 800 22 000

1.31 1.37 1.61

a The molecular weights (Mw, Mn) and molecular weight distribution were analyzed by SEC-MALLS. The degree of substitution (d.s.) of AAM was determined by 1H NMR.

was incubated for 1 h at room temperature with shaking, the absorbance was measured at 400 nm on a TECAN Safire2 plate reader (Tecan Austria GmbH, Austria). Cells. Human embryonic kidney cell line HEK293 was obtained from ATCC (Rockville, MD) and cells between passage numbers 66 and 80 were used in all experiments. The human liver hepatocyte cell line HepG2 was a gift from Professor Edvard Smith (Unit for Molecular Cell Biology and Gene Therapy Science, Karolinska Institute, Sweden). The HEK293 cell line was grown in MEM (Gibco Invitrogen, catalog no. 31095) supplemented with 1 mM nonessential amino acids and 10% fetal bovine serum (FBS, Gibco Invitrogen) at 37 °C under 5% CO2. HepG2 cells were grown in DMEM that contained 4.5 g/L glucose and 3.7 g/L bicarbonate (Gibco Invitrogen, catalog no. 41965) that was supplemented with 10% FBS at 37 °C under 10% CO2. Preparation of Chitosan/DNA Polyplexes. Polyplexes with different amino/phosphate (A/P) ratios and a pDNA concentration of 13.3 µg/mL were prepared by the self-assembly method according to the previously described protocol.39 Briefly, the required amount of sterile filtered chitosan stock solution in MQ grade water (2 mg/mL) was diluted in MQ water, and then a constant amount of pDNA solution (0.5 mg/mL) was added during intense stirring on a vortex mixer (1200 rpm, Heidolph REAX 2000, Kebo Laboratory, Sweden). The complexes were incubated for 30 min at room temperature prior to transfection. Size and Surface Charge Analysis. The size of chitosan/pDNA polyplexes was determined by dynamic light scattering on a Nanosizer ZS apparatus (Malvern Instruments, Malvern, UK). The measurement was taken at an angle of 173° at 25 °C, and each sample was analyzed in triplicate. The size of polyplexes is expressed as the mean diameter (z-average) obtained by cumulant analysis of the correlation function using the viscosity and refractive index of water in calculations. For the measurement of aggregation kinetics, the polyplexes were diluted 1:2 with double-strength phosphate-buffered saline (PBS) (pH 7.2) to

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obtain the isotonic formulations, similarly as in transfection experiments, and the size was repeatedly measured over a period of 120 min. The surface charge (zeta potential) was determined by Doppler velocimetry on the same instrument. In Vitro Transfection Experiments. The cells were seeded in 96well tissue culture plates (Corning Cell-bind, catalog no. 3300) 24 h prior to the transfection experiments to obtain cell confluency of 80-90% on the day of transfection. The polyplexes that were formulated in MQ water were diluted 1:2 by OptiMEM (OptiMEM I, Gibco Invitrogen) that was supplemented with 270 mM mannitol and 20 mM HEPES to adjust the osmomolality and pH. Cells were washed with preheated OptiMEM, and 50 µL of isotonic formulation containing 0.33 µg pDNA was added to each well. After 5 h of incubation, the formulations were replaced by 200 µL of fresh culture medium (MEM, 10% FBS). Culture medium was then changed every second day. The GFP expression was measured in situ by direct recording of fluorescence intensity on a TECAN Safire2 well plate reader. To determine luciferase expression, cells were washed twice with preheated PBS and were lysed with luciferase lysis buffer (Promega, Madison, WI), and the luciferase activity (RLU) was measured on a luminometer (Mediators PhL, Austria). The absolute amount of luciferase expressed was determined from a standard curve prepared with firefly luciferase (Sigma). The total cell protein was determined by the bicinchoninic acid assay (Pierce). Flow Cytometry. Flow cytometry analysis of GFP-expressing cells was conducted on a FACSCalibur system (Becton Dickinson, San Jose, CA), and the data were analyzed by CXP software. Cells were washed twice in PBS, were trypsinized, and were resuspended in ice-cold PBS that was supplemented with 2% FBS. For each sample, 10 000 events were counted, and a dot plot of forward scatter against side scatter was used to establish a collection gate to exclude the cell debris and dead cells, showing reduction in the forward scatter. The GFP-positive cells within this gate were excited by a 488 nm laser line and were detected by the use of a 530/30 nm band-pass filter (FL1). To exclude the autofluorescense, several nontransfected cell populations were run as negative controls to set a gate for GFP-positive cells. MTT Assay. The cytotoxicity of chitosans and their polyplexes with pDNA was evaluated by the MTT method, which measured the intracellular dehydrogenase activity in HEK293 cells. Briefly, HEK293 cells were transfected according to the transfection protocol described above or were exposed to an increasing concentration of free chitosan and PEI. After 5 h of incubation, the complexes or free transfection reagents were removed, cells were washed with preheated OptiMEM, and 20 µL of sterile filtered 5 mg/mL MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide, Sigma-Aldrich) solution in OptiMEM was added. Following 2 h of incubation, the formazan crystals were dissolved by the addition of 200 µL of solubilization solution (M-8910, Sigma). After dissolution, the absorption was measured at 570 nm with a background correction at 690 nm using a TECAN Safire2 plate reader. The instrument was set to zero absorbance using the MTT solution blank. The intracellular dehydrogenase activity of the treated cells was expressed relative to that of the control (untreated) cells. Confocal Microscopy. HEK293 cells were seeded on Lab-Tek chambered cover glasses (Nalge NUNC) and were transfected as described above. After 48 h of incubation at 37 °C under 5% CO2, live cells were examined under a confocal laser scanning microscope (Leica TM TC4D, Leica, Germany). The GFP was excited by a 488 nm laser line, and the emitted light was detected by a standard combination of filters. The images of GFP-positive cells were captured with imaging software (Leica Lite). Statistical Analysis. The experiments were performed on a minimum of two occasions using quadruplicate samples. All data are expressed as mean values ( standard deviation. We investigated statistical differences between mean values by using ANOVA. Differences between means were considered significant at p < 0.05.

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Figure 1. Characterization of the chitosan vectors. (a) Schematic description of the molecular architecture of linear, AAM-substituted (TCO), and self-branched AAM-substituted (SB-TCO) chitosans; (b) SEC-MALLS elution profiles; and (c) solubility of 0.1% chitosans in HEPES-buffered Hanks’ balanced salt solution (HBSS) at 25 °C.

Results Preparation and Solution Properties of Self-Branched Glycosylated Chitosan Oligomers. Nitrous acid depolymerization of fully de-N-acetylated chitosan (FA < 0.002) to the average DPn of 34 yielded chitosan oligomers with a reactive 2,5-anhydro-D-mannofuranose (M) unit at a new reducing end. The reducing ends in depolymerized chitosans are conventionally reduced to avoid the formation of Schiff bases with primary amines.43 Under the reductive alkylation conditions that were used for the substitution of the trisaccharide AAM to the amino group of chitosan, the M units of nonreduced oligomers react with the primary amino groups of chitosan, yielding chitosan with a branched structure (so-called self-branched chitosan). By starting with chitosan oligomers with a reactive M unit at the reducing end and the trisaccharide AAM with the same reactive M unit, we have prepared self-branched glycosylated chitosans that are substituted with GlcNAc containing trisaccharide (SBTCO). Figure 1A shows a schematic illustration of the molecular architecture of the three different chitosan oligomers used in this study: linear, TCO, and SB-TCO. The characterization data including the molecular weights, polydispersity index, and


Figure 2. Physicochemical characterization of chitosan/pDNA complexes. (a) Mean hydrodynamic diameter and zeta potential of polyplexes formed in MQ water at 6 µg/mL pDNA and different amino/ phosphate (A/P) ratios, (b) time-dependent aggregation of polyplexes in PBS, and (c) polyplex stability evaluated by the gel retardation assay in TAE (pH 8.0). pDNA (100 ng) was loaded in each well. Lane 1 contains naked DNA and lanes 2-6, 7-11, and 12-16 contain linear DPn34, TCO, and SB-TCO, respectively, at A/P ratios of 5, 10, 20, 30, and 60.

degree of AAM substitution are presented in Table 1. The comparison of SEC-MALLS elution profiles that is given in Figure 1B shows that self branching of linear oligomers with a DPn of 34 (Mw ) 9.7 kDa) resulted in an increase in the molecular weight of the sample to Mw ) 22 kDa. To investigate the impact of AAM substitution and self branching on solution properties of chitosan, we studied the solubility of the oligomers in the pH range of 6.5 to 7.9. As illustrated in Figure 1C, the AAM substitution considerably improved the solubility of the chitosan. In contrast with the linear oligomer that started to precipitate around pH 7, the TCO remained completely soluble up to pH 7.4, and the SB-TCO remained completely soluble in the whole pH range studied. Physicochemical Characterization of Chitosan/DNA Polyplexes. To examine the effect of chitosan structure on its DNA condensation ability, we prepared a series of polyplexes that was subjected to particle size and surface charge analysis (Figure 2A,B). Despite differences in chitosan structure, all complexes had similar sizes and zeta potentials when reconstituted in MQ water (Figure 2A) with a width of distribution (polydispersity index) of 0.1 to 0.2. The hydrodynamic diameter of the complexes increased from 80 to 150 nm with the


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increasing A/P ratio and thereby the amount of chitosan in the formulation. When the pH and osmomolality of the formulations were adjusted to physiological levels (PBS, pH 7.2, 290 mM), linear polyplexes rapidly aggregated into micrometer-sized structures, whereas TCO polyplexes showed less pronounced aggregation (Figure 2B). Only the polyplexes that were formed by SB-TCO at an A/P ratio of 30 remained stable during the 100 min of assay. For a comparison of the condensation ability of different chitosans, series of chitosan/pDNA polyplexes with A/P ratios from 5 to 60 were exposed to the gel retardation assay. Compared with the linear oligomer, TCO displayed a reduced ability to retard DNA, and even a charge ratio as high as 60 was not sufficient to retain all plasmid (Figure 2C). Polyplexes formed by SB-TCO showed higher physical stability than the linear complexes, forming stable complexes at an A/P ratio of 10. Gene Transfer Efficacy in HEK293 Cells. To establish the relationship between the molecular architecture of chitosan vectors and their gene transfer efficacy, we chose HEK293 cells to be a model cell line. This cell line has been extensively used as an expression tool for recombinant proteins,46 and it is well suited for rapid screening and optimization of the formulations. For a comparison of the gene transfer efficacy of linear, TCO, and SB-TCO, HEK293 cells were transfected with gWiz GFP plasmid as a reporter, and the signal-to-noise ratio (S/N) of GFP fluorescence was recorded after 24, 48, and 72 h, as shown in Figure 3A. Compared with linear and TCO formulations, the SB-TCO showed improved gene transfer efficacy at relatively low A/P ratios. Interestingly, the optimal performance of each chitosan was obtained at different A/P ratios. Whereas transgene expression decreased with increasing A/P ratio from 10 to 30 for linear chitosan, the opposite was true for TCO. SB-TCO showed similar performance at A/P ratios of both 10 and 30. To evaluate the gene transfer efficacy on a single cell level, the GFP-transfected cells were analyzed by flow cytometry. The transfection with SB-TCO resulted in the highest percentage of GFP-positive cells in the population (Figure 3B). At 72 h, 70% of cells transfected with SB-TCO polyplexes at an A/P ratio of 30 expressed GFP compared with 40% transfected with TCO and 20% with the linear oligomer. Besides flow cytometry, the gene delivery efficacy of the chitosans was also examined by confocal microscopy. The images of GFP-expressing HEK293 cells shown in Figure 3C confirm the observed differences in efficacy. From a comparison of the relative amounts of transfected cells at 24, 48, and 72 h, it is apparent that the SB-TCO exhibited a more rapid onset of transgene expression than did linear and TCO. However, the long half life of GFP makes this reporter unsuitable for kinetic studies; therefore, the kinetics of gene expression was examined with luciferase as a reporter. The time course of luciferase gene expression in HEK293 (Figure 4) shows that the SB-TCO clearly outperformed the other two chitosans, and the amount of the expressed protein was approximately 1% of the total protein. After transfection (72 h), the cells transfected with SB-TCO expressed 3 times more luciferase than those transfected with the linear or TCO oligomers. Interestingly, both AAM-substituted chitosans (TCO and SB-TCO) showed different kinetics of luciferase expression compared with linear oligomers; the luciferase expression peaked at 72 h, whereas in the case of linear oligomers, it continued to increase up to 120 h. Next, the compatibility of the chitosan-mediated transfection with serum-containing medium was investigated. All chitosan

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Figure 4. Luciferase gene expression kinetics in HEK293. Cells were transfected with linear polyplexes formed at an A/P ratio of 10 and TCO and SB-TCO polyplexes formed at an A/P ratio of 30. Cells were analyzed for luciferase gene expression at the indicated time points. Data points represent mean ( SD (n ) 4).

Figure 5. Serum compatibility of chitosan formulations. HEK293 cells were transfected by linear (A/P ) 10), TCO, SB-TCO (A/P ) 30), and Exgen (A/P ) 5) in OptiMEM I in the presence or absence of FBS and were analyzed for luciferase gene expression at 48 h. Data points represent mean ( SD (n ) 4). The luciferase gene expression mediated by SB-TCO was significantly higher (*p < 0.05) than that for all other formulations.

Figure 3. Transfection efficacy of chitosan formulations in HEK293 cells. Cells were transfected with complexes of variable A/P ratio at a constant pDNA concentration of 0.33 µg DNA/well. (a) Expression of GFP reporter in HEK293 transfected with linear, TCO, and SBTCO polyplexes at A/P ratios of 10 and 30. The gene expression was measured by recording the fluorescence intensity of the GFP reporter in living adherent cells in situ by a well plate reader at time intervals of 24, 48, and 72 h. The degree of GFP expression is plotted as the signal-to-noise ratio (S/N) relative to nontransfected cells. (b) Flow cytometry analysis of GFP-transfected cells. The percentage of GFP-positive cells in the population transfected with polyplexes formed at A/P ratios of 10 and 30. Data points represent mean ( SD (n ) 4). (c) CLSM image of living GFP-expressing cells 48 h after transfection.

formulations performed well in 10% serum-supplemented medium, and the SB-TCO significantly exceeded the performance of Exgen (Figure 5). In contrast, Exgen-mediated gene transfer was significantly deteriorated by the presence of 10% serum. Effect of pH on Transfection. Bearing in mind the differences in the solubility of chitosans (Figure 1C) at pH > 7, we decided to examine the impact of pH on the gene transfer efficacy of chitosans. Because the ionization degree of chitosan is sensitive to pH changes around a pKa of 6.6, the pH of the transfection medium was varied from 6.8 to 7.9. Figure 6 shows the luciferase gene expression in HEK293 mediated by linear,

Figure 6. Influence of the pH of the formulation of chitosan/pDNA complexes on the luciferase gene expression. HEK293 cells were transfected with linear (A/P ) 10), TCO, and SB-TCO polyplexes (A/P ) 30) in 20 mM HEPES-buffered OptiMEM I adjusted to a pH of 6.8 to 7.9 and were analyzed for luciferase gene expression at 72 h. Data points represent mean ( SD (n ) 4).

TCO, and SB-TCO as a function of pH. Transfection with linear oligomers at pH > 7.2 resulted in a rapid decline in luciferase gene expression, and at pH > 7.4, very low luciferase levels were detected. In contrast, TCO was able to deliver genes at all pHs in the range of 6.8 to 7.9, albeit with reduced efficacy as the pH increased. Increasing the pH from 6.8 to 7.9 during transfection lead to a 75% reduction in luciferase gene expression for TCO compared with only a 40% reduction for SBTCO. Nevertheless, the lowest gene expression that was obtained with SB-TCO (at pH 7.9) was comparable to the highest gene expression that was obtained with TCO (at pH 6.7).


Figure 7. Transfection efficacy of chitosan formulations in HepG2 cells. Cells were transfected with complexes of variable A/P ratio at a constant pDNA concentration of 0.33 µg DNA/well. (a) Luciferase gene expression 48 h post-transfection. The luciferase gene expression mediated by SB-TCO was significantly higher (*p < 0.05) than that for all other formulations. (b) Time course of luciferase gene expression in HepG2. Data points represent mean ( SD (n ) 4).

Gene Transfer Efficacy in HepG2 Cells. For an investigation of whether the self branching of chitosans also leads to improved performance in other cell lines, the human liver hepatocytes HepG2 cell line that expresses the cell surface lectins for GlcNAc was chosen.38 As shown in Figure 7A, both TCO and SB-TCO demonstrated a higher gene expression than linear chitosan in HepG2 cells. SB-TCO mediated 10 and 4 times higher luciferase expression than did linear and TCO, respectively. The time course of luciferase expression after transfection with linear, TCO, and SB-TCO is shown in Figure 7B. Similarly, as for HEK293, the AAM-substituted oligomers showed a more rapid onset of gene expression compared with linear oligomers. The luciferase expression peaked at 48 h and declined more rapidly than it did in HEK293 cells. Cytotoxicity. For an evaluation of the cytotoxicity of free polymers and chitosan/pDNA polyplexes, the activity of mitochondrial dehydrogenase was measured in MTT-based viability assay. Figure 8 shows that none of the chitosans used in this study showed toxic effects up to the concentrations of 0.5 mg/ mL. PEI-based transfection reagents, 25 kDa branched and 22 kDa linear PEI (Exgen), showed comparable dose-dependent toxicities with approximately 50% viability at 0.03 mg/mL. Although transfection with PEI was performed well below this concentration (∼0.009 mg/mL), changes in the cell morphology as well as the loss of cells because of the extensive detachment were often observed, which resulted in poor reproducibility of PEI-mediated transfection.

Discussion In this study, we sought to overcome the traditional limitations of chitosans, such as low solubility and tendency to aggregate,


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Figure 8. Intracellular dehydrogenase activity of HEK293 (a) after transfection with chitosan and PEI polyplexes and (b) after exposure to free transfection reagents, measured by MTT assay. In contrast with Exgen and branched PEI, none of the chitosans or formulations thereof exhibited cellular toxicity. Data points represent mean ( SD (n ) 4), and * indicates p < 0.05.

and to develop a more physiologically compatible chitosan-based transfection reagent with increased gene transfer efficacy and low cytotoxicity. Instead of implementing extensive derivatization procedures that may lead to increased cytotoxicity, we chose a simple molecular tailoring approach to improve the functional properties of chitosan. We hypothesized that the combination of self branching of linear oligomers and AAM substitution would increase the solubility, whereas the former would also compensate for the decrease in the binding affinity of glycosylated oligomers. The low solubility of chitosan under physiological conditions is a major drawback in different biomedical applications. With the exception of highly acetylated chitosans with FA values close to 0.5, which are less efficient as gene carriers,22,40,41 the solubility of chitosans drastically decreases at pHs above 6.0.47 The solubility may be improved to a certain degree by reduction in molecular weight;20,47 however, even an oligomer with a DPn of 34 precipitated above pH 7.0 (Figure 1C). A common approach to increase the solubility and charge density of chitosan at physiological pH is the quarternization of amino groups by trimethylation.25-27 Despite their higher efficacy of gene transfer compared with that of unmodified chitosans, the trimethylated chitosans were shown to be more cytotoxic,25,26 which confirmed the relationship between high charge density and toxicity.5 As shown in this study, the substitution of about 7% of the GlcN units of the fully de-N-acetylated chitosan backbone by the AAM trimer profoundly increased the solubility of the chitosans at neutral pH. The trisaccharide AAM was produced by controlled depolymerization of highly acetylated chitosans.44

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The higher solubility of the TCO at pH > 7 (Figure 1C) may be attributed to difficulties in forming interchain hydrogen bonds due to the increased irregularity in structure, which is similar to the case of highly acetylated chitosan.47 The self branching of the backbone further increased the solubility despite the increase in molecular weight. Despite the differences in molecular architecture, all chitosans in this study compacted DNA into polyplexes of similar sizes and surface charges (Figure 2A). Apparently, the self branching and glycosylation did not interfere with DNA condensation at low pH and ionic strength, where a large entropic contribution of the released counterions drives the self assembly of complexes. This is in agreement with a study that shows that lactosylation of chitosan up to a d.s. of 33% did not influence the particle size and charge of corresponding polyplexes at an A/P ratio of 10.42 The size of polyplexes in the range of 80-150 nm is comparable to these lactosylated complexes42 but is below the size of chitosan/DNA complexes reported elsewhere,40,41 which possibly reflects lower ionic strength and the use of chitosan samples with lower polydispersity. The high positive surface charge gives rise to an electrostatic barrier that prevents aggregation, and the polyplex solutions remained stable for at least 1 week (data not shown). Following the exposure of preformed chitosan/pDNA polyplexes to conditions resembling the physiological situation (PBS, pH 7.2), the glycosylated chitosans displayed improved colloidal stability compared with unsubstituted counterparts, which is in agreement with previously reported data.38,42 The polyplexes formed by SB-TCO demonstrated the highest colloidal stability, which is probably due to the combination of the sterical hindrance effect and an increased chain length, similarly as observed for trehalose-based polymers.9 Clearly, the combination of AAM substitution and branching of the backbone allowed for the preparation of sterically stabilized nanoparticles. The physical stability of the polyplexes is critically dependent on the number of protonized primary amino groups and thereby the degree of cooperativity of interactions.32,36,39 As expected, the random substitution of the uncharged AAM ligand to the chitosan oligomer reduced the affinity for DNA, leading to weaker DNA polyplexes. To compensate for the loss of affinity, higher amounts of TCO had to be applied to form stable polyplexes (Figure 2C), similarly as observed for chitosans with an increasing degree of acetylation.36 The self branching of the chitosan backbone counterweighed this effect because of the increase in the number of charges following the increase in molecular weight, and SB-TCO polyplexes retained DNA comparable with that of linear unsubstituted chitosan. The gene transfer experiments in HEK293 using GFP as a reporter revealed that the SB-TCO outperformed the linear and TCO formulations in terms of the percentage of transfected cells and the amount of GFP produced (Figure 3). Although a direct comparison of transfection results among different laboratories is difficult, the percentage of HEK293 cells transfected by linear oligomer is similar to results reported by Lavertu.40 A comparison of the transfection patterns that were obtained from flow cytometry (Figure 3B) with fluorescence intensity levels (Figure 3A) makes it apparent that the correlation between the percentage of transfected cells and the amount of transgene decreased at later time intervals (48 and 72 h) because of intracellular accumulation of GFP. Clearly, the long half life of the GFP makes this reporter unsuitable for kinetic studies. Therefore, the same plasmid construct containing luciferase, a protein with an intracellular half life of 2 to 3 h, was used as a reporter gene for kinetic studies and for the quantification of

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the amount of transgene produced. The rapid luciferase expression mediated by the SB-TCO indicates an efficient release of pDNA and intracellular trafficking to the nucleus. In comparison, the slow but steady increase in luciferase levels in cells transfected by linear oligomers may suggest that the release of DNA is less efficient compared with that of the AAM oligomers. It was previously reported that dissociation of DNA from chitosan/DNA complexes occurred at different locations in the cell depending on the DNA retention capability of the chitosan used.41 However, SB-TCO and linear oligomers had similar DNA release profiles in the gel electrophoresis experiment. This may suggest that the linear and SB-TCO polyplexes differ in some stage in the intracellular processing of complexes, and this hypothesis is currently under investigation. In most cases of in vitro transfection using polymer and lipidbased vectors, the transfection efficacy is reduced in the presence of serum, and serum-free medium (OptiMEM) is traditionally employed in cell transfection protocols. The binding of serum protein to polyplexes is thought to be an important factor in limiting bloodstream circulation and restricting access to target tissues.48 The compatibility of chitosan complexes with serum (Figure 5) is therefore an important advantage for their use in vivo and in vitro. Recently, trehalose-based polymers were also shown to exhibit serum compatibility,10 suggesting that this feature may be shared by various glycopolymers. Because of the pH-dependent degree of ionization of chitosan, the pH of the formulation and during the transfection experiment is undoubtedly one of the most important parameters for successful transfection. Transfection with chitosan-based reagents in vitro has been typically performed in acidic media.35,39 For reasonable transfection to be achieved in vitro with chitosan oligomers of 4 to 6 kDa, acetate buffer (pH 5.5) had to be employed.39 Similarly, transfection at pH 6.5 increased luciferase expression compared with that performed at pH 7.1.40 Whereas the use of acidic media is acceptable (e.g., for lung administration of aerosols in vivo), it may contribute to irritation and cytotoxicity in other circumstances, and the high gene delivery performance of SB-TCO at high pH values such as pH 7.8 is therefore particularly encouraging. Because the pKa value of the substituted secondary amino group is 5,44 this effect cannot be explained by the higher charge density of SB-TCO. Most likely, the pH tolerance is a consequence of increased solubility and colloidal stability at high pH. The poor performance of linear oligomers at pH > 7.2 confirms that when standard transfection protocols are employed, the use of conventional chitosans as gene delivery vectors is problematic. It has been previously reported that the presence of AAM ligand in TCO resulted in increased uptake because of the targeting of GlcNAc binding lectins in HepG2 cells.38 Also, in this study, the AAM-containing chitosans possessed higher gene transfer efficacy compared with AAM-deficient oligomers. However, the presence of the ligand alone cannot explain the significantly increased luciferase expression levels after transfection with SB-TCO. Again, the improved properties of formulation may account for increased efficacy. The time course of gene expression revealed that the onset of gene expression is even faster than that in HEK293; however, the transgene production also declined more rapidly. The comparison of the luciferase levels makes it apparent that transgene production is about 10 times higher in HEK293 than in HepG2 cells. Unfortunately, information about the transfection efficacy on a single cell level could not be obtained for HepG2 cells because this cell line turned out to be difficult to resuspend as a single cell suspension.


Conclusions The SB-TCO chitosan that was developed in this study showed several advantages over linear chitosan oligomers, TCO, or PEI-based transfection reagents. It was fully soluble at a neutral pH and formed polyplexes that exhibited high colloidal as well as physical stability without impairing the intracellular release of DNA. In addition, the gene transfer with SB-TCO chitosan tolerated the presence of serum during transfection as well as a broad range of physiological pH values. The rapid onset of transgene expression in both cell lines suggests efficient DNA release and intracellular processing compared with linear and TCO samples. The high percentage of transfected cells as well as the high amounts of transgene produced indicate that SB-TCO possesses high gene delivery potential in vitro. All chitosan formulations had significantly better toxicity profiles than did linear and branched PEI. Consequently, this study challenges the efficacy-toxicity correlation in gene delivery and shows that efficient transfection may be achieved without acute cytotoxic effects. Acknowledgment. This work was supported by a post doc fellowship from Norwegian University of Science and Technology to S.P.S. and a grant from Novamatrix/FMC Biopolymer AS (Oslo, Norway). Supporting Information Available. 1H NMR spectra of linear, TCO, and SB-TCO chitosans. This material is available free of charge via the Internet at http://pubs.acs.org.

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