N-Alkylated Chitosan as a Potential Nonviral Vector for Gene

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Bioconjugate Chem. 2003, 14, 782−789

N-Alkylated Chitosan as a Potential Nonviral Vector for Gene Transfection Wen Guang Liu, Xin Zhang, Shu Jun Sun, Guang Jie Sun, and Kang De Yao* Research Institute of Polymeric Materials, Tianjin University, Tianjin 300072

Dong Chun Liang, Gang Guo, and Jing Yu Zhang Tianjin Medical University, Tianjin 300070. Received September 15, 2002; Revised Manuscript Received February 10, 2003

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The selfaggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the selfaggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (() of 1/1; ACS/DNA complexes were formed at a lower charge ratio (() of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoylsn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C2C12 cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.

INTRODUCTION

In the past decades, chitosan (CS), a naturally occurring linear cationic polysaccharide, has been widely employed as a drug delivery system, wound dressing, anticoagulants, and scaffolds for tissue engineering owing to its biocompatibility, biodegradability, and low toxicity (1-4). Recently, increasing attention has been paid to using chitosan and its derivatives as nonviral vectors for gene transfection (5-9). For example, Mumper (10) was the first to propose to deliver a gene into a cell using chitosan as a vector. The particle sizes of chitosan/DNA complexes ranging from 150 nm to 600 nm were found to depend on the molecular weight of the chitosan (108540 kDa) used, but not on buffer composition. The small plasmid/chitosan nanoparticles (200-300 nm) were prepared by Roy et al. (11) and were shown to transfect HEK 293 cells (human embryonic kidney cells) in vitro at a lower level than with a Lipofectamine formulation. Borchard et al. (12) synthesized trimethylated chitosan (TMO) from oligomeric chitosan, and the obtained TMOs were used to condense RSV-R3 luciferase plasmid to form complexes (chitoplexes), which were investigated for their ability to transfect COS-1 and Caco-2 cell lines in the presence and absence of fetal calf serum (FCS). It was demonstrated that the transfection efficiency of the chitoplexes was not affected by FCS, whereas the trans* To whom correspondence should be addressed. E-mail address: [email protected].

fection efficiency of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]N,N,N-trimethylammonium sulfate) lipoplexes was decreased. Moreover, cells remained 100% viable in the presence of chitosan oligomers and TMOs; while the viability of DOTAP-treated cells decreased to ∼50% in both cell lines. Sato et al. found that chitosan of 15 and 52 kDa obviously enhanced the transfection efficiency of luciferase plasmid (pGL3) into tumor cells. Furthermore, the transfection efficiency of chitosan/pGL3 complexes at pH 6.9 was higher than that at pH 7.6, suggesting chitosan might selectively transfect tumor in the low pH environment, but not be uptaken into normal cells. It was also observed that chitosan was resistant to serum, and while cationic liposome-associated gene expression was inhibited by serum. This resistance to serum implied that chitosan might be an efficient gene vector in vivo. Although in some cases, the uptake of chitosan/DNA nanoparticles appears to occur even in the absence of any ligand-receptor interaction, to allow for the targeted gene trafficking into specific cells, chitosan needs to be modified with various biospecific ligands. Cho’s group (13, 14) developed galactosylated chitosan-graft-dextran and galactosylated chitosan-graft-PEG vectors. Galactose groups were chemically bound to chitosan for livertargeted delivery and dextran or PEG was grafted for enhancing the complex stability in water. These systems could efficiently transfect HepG2 cells expressing asialoglycoprotein receptor (ASGR) which specifically recognizes the galactose ligands on chitosan. Leong’s group

10.1021/bc020051g CCC: $25.00 © 2003 American Chemical Society Published on Web 06/13/2003

N-Alkylated Chitosan as Potential Nonviral Vector

reported that binding transferrin or KNOB (C-terminal globular domain of the fiber protein) onto the surface of chitosan/DNA complex could improve gene expression level in HEK293 and HeLa cells to a different degree (15). The advantage of chitosan-based vectors lies in not only getting away from the cytotocixity problems that are inherent in most synthetic polymeric vehicles but also in its unique capability of transcellular transport (16, 17). However, as shown with other polycations/DNA complexes, chitosan/DNA complexes are formed by electrostatic interaction between primary amino groups and phosphate groups, which is strong enough to resist DNA unpacking within cell to a certain degree. Okano (18), Sato (19), and Kabanov (20) all reported that the incorporation of hydrophobic moieties could considerably increase the transfection efficiency. In addition, by theoretical calculations Kuhn and Levin found that for sufficiently hydrophobic amphiphilies, a charge neutralization, or even a charge inversion of the DNA-amphiphile complexes could be achieved with rather low concentration of cationic amphiphile (21). To the best of our knowledge, to date, research work on the influence of hydrophobicity of chitosan on the properties of chitosan/DNA complexes and on the transfection efficiency of chitosan-based vectors is unavailable in the literature. In this work, we synthesized a series of alkylated chitosan (ACS) derivatives from alkyl bromide and investigated the stability of ACS/DNA complexes; an attempt was made to elucidate the mechanism of the interaction between ACS and mimic cell membrane, dipalmitoyl-sn-glycocerol-3-phosphocholine (DPPC). The CS and ACSs were used as vectors for gene transfection, and the effects of hydrophobicity of the side alkyl chain on the transfection activity were explored. EXPERIMENTAL SECTION

Materials. Chitosan (CS) (Mν ) 50 000, degree of deacetylation ) 99%), DNase I was supplied by Sigma Chemical Company (St. Louis, MO). Pyrene obtained from Aldrich was purified by repeated recrystallizations from absolute ethanol. Alkyl bromides were analytically pure. pcDNA 3.1/CAT plasmid encoding chloramphenicol acetyltransferase (CAT) was purchased from Ivitrogen Co. Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in powder form was obtained from Fluka Co. and was used as received. Ethidium bromide and HEPES were purchased from Sigma. DMEM (Dubelco’s Modified Eagle Medium) powder was purchased from Gibco Co. Fetal bovine serum (FBS) was provided by Hyclone Co. Synthesis of N-Alkylated CS. The synthetic method of N-alkylated chitosan has been reported in our previous paper (22). Briefly, 2 g of CS was added into 40 mL of 2-propanol/4 N sodium hydroxide solution and stirred at 70 °C for 30 min. The alkyl bromide was added dropwise to the mixture and allowed to react for 4 h, and then the reaction mixture was centrifuged. The obtained precipitate was washed with ethanol and then dried at vacuum to obtain the alkylated chitosan derivatives. The resultant CS derivatives from butyl bromide, octyl bromide, dodecyl bromide, and hexadecyl bromide were denoted as 4-, 8-, 12-, and 16-CS, respectively. The resultant alkylated chitosan derivatives were dialyzed for 3 days using Cellu SepH1 membrane (MWCO ) 12 000) against water. The degree of substitution was determined by traditional potentiometric titration. Fluorescence Measurement. Chitosan and alkylated chitosans were dissolved in 0.1 M of acetic acid, and the concentrations of sample solutions were varied from

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1.0 × 10-4 to 5 mg/mL. Steady-state fluorescence measurement was performed on a SPEX FL212 Spectrofluorometer. For the determination of intensity ratio of the first to the third highest energy bands in the emission spectra of pyrene, the sample solution containing pyrene (8.0 × 10-7 mol/L) was excited at 338 nm, and the emission spectra were recorded in the range of 350-420 nm at the scan rate of 15 nm/min. The slit openings for excitation and emission were set at 2.0 and 0.5 nm, respectively. The spectra were accumulated with an integration time of 5 s/nm. Dynamic Light Scattering Determination (DLS). Dynamic light scattering measurement was carried out with an argon ion laser system tuned at 514 nm. The solutions of chitosan derivatives were filtered through a 0.5-µm filter (Millipore) directly into a freshly cleaned 10 mm-diameter cylindrical cell. The intensity of autocorrelation was measured at a scattering angle (θ) of 90° with a Brookhaven BI-9000AT digital autocorrelator at room temperature. When the difference between the measured and the calculated baselines was less than 0.1%, the correlation function was accepted. The mean diameter was evaluated by the Stokes-Einstein relationship. CS/DNA and ACS/DNA Complex Formation. CS was dissolved in 0.1 M sodium acetate buffer to form a solution of 1 mg/mL, and a DNA solution of 0.1 mg/mL was formed in the same way. Chitosan/DNA complexes at various charge ratios were prepared by mixing chitosan solution with DNA solution, vortexing for 15 s and incubated for 30 min at room temperature. Likewise, ACS/DNA complexes were formed. The complex formation was confirmed by electrophoresis on an 1.0% agarose gel with Tris-acetate (TAE) running buffer at 100 V for 30 min. DNA was visualized with ethidium bromide. DNase Resistivity. CS/DNA or ACS/DNA complexes were placed into 2 mL of 10 mM PBS buffer containing 5 mM MgCl2 at 25 °C. To this solution was added 10 units of DNase I, and the optical density of the solution at 260 nm was recorded for 1 h. Differential Scanning Calorimetry Determination. The interactions between DPPC and chitosan or its derivatives were determined with thermal analysis method reported by Fang et al. (17). Differential scanning calorimetry (DSC) scans were carried out on a PerkinElmer DSC-7 calorimeter. A certain amount of DPPC and chitosan mixed in 25 µL of PBS buffer was hermetically sealed in an aluminum pan. For each DSC run, 25 µL of plain PBS buffer was loaded in the hermetic pan and used as a blank reference. Prior to scanning, the aluminum pan loaded with sample was equilibrated at 60 °C with constant vortexing for at least 2 h in order to disperse DPPC and DPPC/CS mixtures in aqueous solution. Heating or cooling scans were recorded in the range of 25-50 °C at a scan rate of 5 °C/min. In the same manner the thermograms of ACS/DNA mixtures were recorded. AFM Imaging. Two micrograms of DPPC and 0.5 mol % of chitosan were mixed together in PBS buffer and equilibrated at 60 °C with vortexing for 3 h, and then 20 µL of mixture was deposited onto a freshly cleaved mica disk. Likewise, DPPC and DPPC/ACS mixture samples were prepared. To visualize the morphological variation of DPPC membrane induced by CS/DNA or ACS/DNA complexes, 1% of DPPC was dissolved in chloroform, 20 µL of diluted solution was deposited onto a freshly cleaved mica to form a homogeneous membrane, and then 15 µL of CS/DNA (( ) 1/1) or ACS/DNA (( ) 1/4) complex solution was

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deposited onto the DPPC-precoated mica. All the deposited solutions were dried at room temperature. The imaging was conducted on an Atomic Force Microscopy (Nanoscope a system, Digital Instruments, Inc. Santa Babara, CA) in tapping mode, with 512 512 data acquisition at a scan speed of 1 Hz at ambient conditions. Cell Culture and Transfection. C2C12 cells, a mouse skeletal muscle cell line, which have been shown to be a suitable host for stable transfection experiments of exogenous DNA (23), were seeded at a density of 5 × 105/ mL on 24-well microplates in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% fetal bovine serum at 37 °C under a 5% CO2 atmosphere. When the cells were grown to half confluency, the culture media were extracted, and rinsed with serum-free DMEM. The prepared chitosan/DNA complexes were diluted with serum-free DMEM and added into the corresponding wells (5 µg DNA/well). After 1 h incubation, the complexes were removed, and the culture media were replaced by fresh serum-containing media and incubated for additional 48 h at 37 °C under a 5% CO2 atmosphere. Then the cells were lysed with PBS solution containing 1 NP40 and 1 mmol/L PMSF and subjected to three cycles of deep freezing and thawing. The CAT concentration was determined with enzyme-linked immunosorbent assay (ELISA) method. The OD280 and OD260 of cell lysis solutions were measured and diluted with PBS to ensure that each sample contains the same protein concentration. The samples were added into 96-well microplates and left at 4 °C overnight. Then the solution was decanted, and 150 µL of 4% of degreased milk powder was added, which was allowed to react at 37 °C for 2 h. After the degreased milk powder was removed, the wells were washed with PBS three times, and 100 µL of rabbitanti CAT IgG was added into each well, which was left at 37 °C for 1 h and followed by rinsing with 0.2% TweenPBS. Then 100 µL of goat-anti rabbit IgG-HRP was added into each well. After incubation at 37 °C for 1 h, 100 µL of TMB was added, and the plate was kept in dark for 10 min. Finally 100 µL of 2 M sulfuric acid was added to terminate the reaction, and the OD values were measured at wavelength 450 nm by plate-reader of Labsystem. The naked DNA was used as a control. Each transfection experiment was carried out in triplicate. RESULTS AND DISCUSSION

Characteristics of N-Alkylated Chitosans. The characterization of N-alkylated chitosan has been discussed in reference (22). The substitution degrees (SD) of alkylated chitosan determined by potentiometer titration are ca. 22.1%; thus, the influence of SD is negligible. In studying the formation of aggregates from hydrophobically modified polyelectrolyte in aqueous solution, pyrene is generally used as a molecular probe, and the variation in the ratio of intensity of first (373 nm) to third (383 nm) vibronic peaks I1/I3, the so-called polarity parameter, is quite sensitive to the polarity of microenvironment where pyrene is located (24). Thus, the change of I1/I3 can characterize the formation of self-aggregation. Figure 1 exhibits the change of I1/I3 value as a function of concentration of chitosan or alkylated chitosans. For 4-CS and 12-CS, at lower concentrations, I1/I3 values remain nearly unchanged. Further increasing concentration, the intensity ratios start to decrease, implying the onset of self-association from alkylated chitosans. The critical aggregation concentration (cac) is determined by the interception of two straight lines. The cac values of alkylated chitosans are listed in Table 1. From the table, it can be seen that the cac of 12-CS is lower than that of

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Figure 1. Variation of intensity ratio (I1/I3) versus the concentration of chitosan or its derivatives. Table 1. Characteristics of Alkylated Derivatives sample

critical aggregation concentration, cac × 103, mg/mL

mean diameter (nm)

4-CS 12-CS

7.24 2.33

261.7 ( 30.1 346.3 ( 51.8

4-CS. A most probable reason is that the elongation of alkyl side chains increases the hydrophobicity, rendering the self-aggregation more prone to occur. It is noted that no aggregation occurs for chitosan in the whole range of concentrations. Whereas Philippova (24) argues that there exist hydrophobic domains inherent to chitosan. In acetic acid solution, chitosan and its derivatives are positively charged, and thus electrostatic repulsion weakens the hydrophobic attraction to a certain degree. The substitution degree of chitosan used by Philippova is 88%, i.e., there still exist parts of acetyl groups which are hydrophobic in nature. These hydrophobic moieties can compete with electrostatic repulsion and lead to the hydrophobic aggregates. In contrast, the chitosan used by us is almost completely deacetylated. Therefore, the hydrophobicity of acetyl can be neglected. DLS data demonstrate that the mean size of selfaggregates from 12-CS is larger than that of 4-CS (Table 1), suggesting the elongation of hydrophobic side-chain facilitates the growth of the hydrophobic core of polymeric aggregates. Formation of CS/DNA and ACS/DNA Complexes. The electrophoretic retardation bands on agarose gels can characterize the formation of the two oppositely charged polyelectrolyte partners since the neutralization and/or increase in molecular size of complex results in the complete retardation of DNA migration toward the anode in the electric field (25). Figure 2 shows the agarose gel electrophoresis results for CS/DNA and ACS/DNA complexes. One can see that a complete retardation occurs at charge ratio(() of 1/1 for CS/DNA complex, indicating the formation of complex. While for 4- CS, 8-CS, 12-CS, and 16-CS, complexes are formed at a relative lower charge ratio of chitosan to DNA, 1/4. It is obvious that not only electrostatic interaction but also hydrophobic interaction contributes to the complex formation between DNA and chitosan derivatives. The incorporation of hydrophobic moieties might cause a cooperative binding transition of alkyl chitosan. Along with the binding of ACS onto the phosphate groups of DNA, polyion charge neutralization occurs, and the bound ACSs facilitate the binding of subsequent ones via favorable hydrophobic interactions of alkyl side chains. At low concentration of ACS, there might be a discontinuous rising in the binding



Figure 2. Electrophoresis of CS/DNA (a), 4-CS/DNA (b), 8-CS/DNA (c), 12-CS/DNA (d), and 16-CS/DNA (e) complexes on an agarose gel. Lane 1: plasmid DNA; lane 2: +/- ) 1/10; lane 3: 1/1; lane 4: 1/4; lane 5: 1/1; lane 6: 2/1; lane 7: 4/1; lane 8: 8/1; lane 9: 10/1.

Figure 3. Variation in the optical density of DNA at 260 nm in PBS buffer containing DNase I.

of ACS, i.e., a cooperative binding (26). Thus, even below the stoichiometric point of neutralization, the charges on the backbones of DNA are shielded by the bound hydrophobic alkyl chains. For ACS/DNA complexes, at charge ratio (( ) 1/4), the migration of DNA has been completely hindered. Therefore, a small amount of alkylated chitosan derivatives can condense DNA. Kuhn et al. (21) argued that lowering the amount of cationic amphiphiles could reduce the risk of unnecessary medical complications. But as a vector, the amphiphile must protect DNA from nuclease degradation. To examine whether chitosan itself or the incorporated alkyl side chain can protect DNA from the attack of nuclease, we recorded the variation in optical density of the CS/DNA (( ) 1/1) and ACS/DNA (( ) 1/4) complexes in PBS buffer containing DNase I at 260 nm (Figure 3). The charge ratio of components is selected as 1/1 and 1/4 for CS/DNA and ACS/DNA complexes, respectively, taking account of that at these two ratios, the complexes between DNA and CS or ACS are just formed. As shown in the figure, the OD260 values of naked DNA increase sharply with time increasing, suggesting DNA has been degraded. In contrast, the OD260 values of DNA entrapped in the complexes remain lower values, and is nearly unchanged after 10 min. As expected, the complex

formation between DNA and chitosan can reduce the activity of DNase. It is noted that although the selected charge ratio of ACSs to DNA is lower relative to that of CS/DNA complex, an equal inhibition effect was observed for ACSs. Thus it is reasonable to consider that a small amount of alkylated chitosan can play the same shielding role as chitosan. In our previous work (27, 28), we have employed AFM to observe that the dodecylated chitosan offers a good protection for DNA, which is surmised that the hydrophobic alkyl chains incorporated lowers the permeability of DNase. Interactions of CS or ACS with DPPC. Figure 4 displays the DSC heating thermograms of DPPC/CS and DPPC/ACS mixtures at different mole fractions of chitosan or its derivatives in PBS buffer. It is clearly seen that two endothermic transition peaks appear around 33 and 41.5 °C, revealing a typical gel-liquid crystalline transition occurs in DPPC multilamellar vesicles(MLV) (16). Along with the increase in the contents of CS or ACS, the peak height of main transition at 41.5 °C is considerably decreased, and the peaks are slightly broadened, an indication that the heterogeneity of membrane is enhanced. Figure 5 exhibits the DSC cooling thermograms of DPPC/CS and DPPC/ACS mixtures. Pure DPPC and all the mixtures have a major exothermic peak around 37.5 °C. For DPPC/CS and DPPC/ACS mixtures, a new exothermic transition at 30.5 °C is split from the thermograms, which implies that the incorporation of chitosan or its hydrophobic derivative results in the phase separation of DPPC. Table 2 lists the calorimetric enthalpy of DPPC bilayer determined at different mole fractions of chitosan or its derivatives. From the table, one can see that increasing the contents of CS or ACS leads to the decrease in enthalpy of DPPC, demonstrating the addition of CS or ACS suppresses the cohesive interactions between adjacent DPPC molecules. Furthermore, with the elongation of alkyl side chain, the reduction of enthalpy is more evident. Fang et al. (17) argues that not only long-range repulsion between choline and cationic amines, but also hydrophobic interaction between chitosan and DPPC, induces bilayer destabilization. It is necessary to emphasize that the chitosan used in our experiment is fully


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Figure 4. DSC thermograms of DPPC/CS mixtures at different mole fractions of CS or ACS in PBS during heating. (a): DPPC/CS; (b): DPPC/4-CS; (c): DPPC/12-CS; (d): DPPC/16-CS.

Figure 5. DSC thermograms of DPPC/CS mixtures at different mole fractions of CS or ACS in PBS during cooling. (a): DPPC/CS; (b): DPPC/4-CS; (c): DPPC/12-CS; (d): DPPC/16-CS.

deacetylated; so the hydrophobicity from acetyl moieties can be neglected. It is obvious that the strong hydrophobic interactions are originated from alkyl side chain introduced. The longer side chains might penetrate the hydrophobic core of DPPC bilayer, and consequently enhance the disruptions of DPPC. In addition, we find no irreversible transition of DPPC during its heating and cooling as shown in the table.

Figure 6 exhibits AFM images of DPPC MLV, DPPC/ CS and DPPC/12-CS mixtures. For pure DPPC, the lamellar structure of stacked bilayer with a mean diameter of 4 µm is formed. Upon associating with chitosan, the fusion of bilayer is observed, and the surface of DPPC is indented to some extent. Similarly, the incorporation of 12-CS leads to the fusion of vesicles, and the mean size increases slightly compared to that of pure DPPC.



Table 2. Calorimetric Enthalpy of DPPC Bilayer Determined at Different Mole Fractions of Chitosan or Its Derivativesa mole fraction (%)

CS ∆Hen ∆Hex

4-CS 12-CS 16-CS ∆Hen ∆Hex ∆Hen ∆Hex ∆Hen ∆Hex

0.5 1.0 5.0 DPPC

6.38 6.41 4.61 4.58 1.78 1.65 11.87 12.21

5.64 3.56 1.55

5.53 3.47 1.76

5.79 3.58 1.02

5.18 3.58 1.48

4.67 3.01 1.27

4.71 3.14 1.20

a ∆H is the endothermic enthalpy and ∆H is the exothermic en ex enthalpy. The unit of calorimetric enthalpy is J/g.

Figure 7. AFM images of naked DNA (a), the complexes of CS/DNA (b), and 12-CS/DNA (c) deposited on DPPC membranes.

Figure 6. AFM images of DPPC MLV (a), DPPC/CS (b), and DPPS/12-CS (c) mixtures.

The AFM images obtained reveal that chitosan or its alkyl derivative can strongly induce the disruption of the integrity of DPPC membrane.

To mimic the interactions between vectors with cell membrane, the DPPC membrane was first formed on mica substrates, onto which naked DNA, CS/DNA, and ACS/DNA complexes were deposited. AFM was used to examine the variation in the topological structure of DPPC (Figure 7). As shown in Figure 7a, the naked DNA molecules occur to aggregate on the surface of DPPC. The size of DNA varies from 100 to 150 nm, and the average height is about 13 nm. The aggregates of DNA are merely absorbed onto the surface of DPPC, and no perturbation of DPPC membrane was observed. In contrast, CS/DNA (Figure 7b) and 12-CS/DNA (Figure 7c) complexes lead to the strong perturbations of DPPC membrane. CS/DNA complex evolves into circular features with size ranging from 250 to 330 nm. It can be also observed that part of


Figure 8. Transfection efficiency of pc DNA 3.1 plasmid encoding CAT mediated by CS and ACS.

the complex particles are aggregated and form larger domains. Comparatively, 12-CS/DNA complex forms an irregular lump with larger size, and the aggregation becomes more evident. A closer inspection of the pictures reveals that it is hard for naked DNA to enter the cell membrane due to electrostatic shielding, whereas chitosan or chitosan derivatives facilitate DNA entry into cell membrane due to the perturbation caused by electrostatic and hydrophobic interactions. Cell Transfection. The transfection efficiency was evaluated in C2C12 cell lines using pc DNA 3.1 plasmid encoding chloramphenicol acetyltransferase (CAT). Figure 8 shows the transfection efficiency of C2C12 cells obtained with CS and ACSs. The OD450 of the control group (naked DNA) is merely 0.136; in contrast, chitosan increases the transfection efficiency 4-fold. The transfection level is further increased upon elongating the alkyl side chain. For 4-CS, the transfection efficiency is increased about 5-fold. 8-CS, 12-CS, and 16-CS yields even higher transfection efficiencies with 7-fold increase compared to that of DNA alone. Nonetheless, the transfection efficiency levels off after the number of carbons in the side chain exceeds eight. Objectively speaking, the experimental results available in the present literature on the effects of complex size upon the transfection efficiency are inconsistent. Several reports support the use of ∼l00 nm diameter of complex particles (24, 29), and several studies demonstrate that submicrometer or micrometer of complex particle yields higher transfection efficiency (30, 31). In studying the effects of the aggregation states on the intracellular uptake of Pluronic copolymer, Muniruzzaman et al. (32) proposed that Pluronic unimers enter into cells via a simple diffusion, whereas the Pluronic micelles are internalized through endocytosis. As shown in the aforementioned AFM images, the size of CS/DNA complex is in the range of several hundreds of nanometers and the ACS/DNA complexes form a larger size. Before complexing with DNA, the alkylated chitosans selfaggregate, and when the self-aggregates of ACS complex with DNA, a larger size of complexes tend to be generated. AFM images also imply that the complexes reaggregate onto the cell surface before they enter into cell membrane. From the analysis above, the complexes of CS/DNA and ACS/DNA are ferried into the cell mainly via endocytosis. Moreover, the increase in hydrophobicity of chitosan derivatives raises its ability to destabilize the membrane, which facilitates DNA entry into cell. However, neither high cellular uptake, membrane destabilizing activity, endosomal escape, nor nuclear localization alone is adequate for a high transfection efficacy, which

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results from complex interplay of various factors, one of which, the unpacking of complex from carrier, is the final limiting step of gene delivery (33). Chitosan has been demonstrated to possess a substantial buffering capability so that it can escape from endosome, avoiding the hydrolysis of lysosomal nuclease (34). In preparing the alkyl chitosan derivatives, only small fractions of primary amino groups were substituted, so most of amino moieties still play a role in absorbing protons of endosomal vesicles. What is more important is that aside from the ionic interactions, there are hydrophobic interactions between alkyl chitosans and DNA, and moreover a small amount of alkyl chitosans can condense DNA, which weakens the strong ionic interaction, promoting the unpacking of DNA from its cargo. Therefore, the incorporation of alkyl side chain enhances the transfection efficiency by comprehensive factors. CONCLUSION

Alkylated chitosans (ACSs) self-aggregate in acetic acid solution, and chitosan (CS) with 99% deacetylation degree does not aggregate due to the strong electrostatic repulsion. The complex formation between ACS and DNA requires a relatively smaller amount of ACS compared to CS due to hydrophobic interactions. CS and ACS can cause the fusion of DPPC multilamellar vesicles and the membrane destabilization. Furthermore, introducing alkyl side chains results in a more evident alteration in the topological structure of DPPC. The transfection efficiency of plasmid-encoding CAT mediated by CS and ACS into C2C12 cell lines is dependent on the hydrophobiciy of chitosan. Upon lengthening the alkyl side chain, the transfection efficiency is raised and levels off after the number of carbons in side chain exceeds 8. The higher transfection efficiency of ACS is presumably due to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers, which is from the hydrophobicity-induced weakening of electrostatic attractions between cargo and carriers. ACKNOWLEDGMENT

The authors are indebted to the financial support from National Natural Science Foundation of China (Grant 50233020), Postdoctoral Foundation (Grant 2002031165), and Joint Research Project of Tianjin-Nankai Universities from the Ministry of Education, China. LITERATURE CITED (1) Thanou, M., Verhoef, J. C., and Junginger, H. E. (2001) Oral drug absorption enhancement by chitosan and its derivatives. Adv. Drug Delivery Rev. 52, 117-126. (2) Mi, F. L., Shyu, S. S., Wu, Y. B., Lee, S. T., Shyong, J. Y., and Huang, R. N. (2001) Fabrication and characterization of a spongelike asymmetric chitosan membrane as a wound dressing. Biomaterials 22, 165-173. (3) Vongchan, P., Sajomsang, W., Subyen, D., and Kongtawelert, P. (2002) Anticoagulant activity of a sulfated chitosan. Carbohyd. Res. 337, 1233-1236. (4) Madihally, S. V., and Matthew, H. W. T. (1999) Porous chitosan scaffolds for tissue engineering. Biomaterials 20, 1133-1142. (5) MacLaughlin, F. C., Mumper, R. J., Wang, J., Tagliaferri, J. M., Gill, I., Hinchcliffe, M., and Rolland, A. P. (1998) Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. J. Controlled Release 56, 259-272. (6) Sato, T., Ishii, T., and Okahata, Y. (1999) In vitro gene delivery mediated by chitosan. Proc. Int. Symp. Controlled Release Bioact. Mater. 26, 803-804.

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N-Alkylated Chitosan as a Potential Nonviral Vector for Gene

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