Urea Aqueous

Cellulose (cotton linter pulp) was supplied by Hubei Chemical Fiber Group Ltd. ... an elemental analyzer (CHN-O-Rapid, Foss Hera us GmbH, Hanau, Germa...
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Biomacromolecules 2008, 9, 2259–2264

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Homogeneous Quaternization of Cellulose in NaOH/Urea Aqueous Solutions as Gene Carriers Yongbo Song, Yunxia Sun, Xianzheng Zhang, Jinping Zhou,* and Lina Zhang Department of Chemistry, Wuhan University, Wuhan 430072, China, and Key Laboratory of Biomedical Polymers, Ministry of Education, Wuhan University, Wuhan 430072, China Received April 20, 2008; Revised Manuscript Received June 1, 2008

Quaternized celluloses (QCs) were homogeneously synthesized by reacting cellulose with 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) in NaOH/urea aqueous solutions. The structure and solution properties of the QCs were characterized by using elemental analysis, FTIR, 13C NMR, SEC-LLS, viscometer, and ζ-potential measurement. The results revealed that water-soluble QCs, with a degree of substitution (DS) value of 0.20-0.63, could be obtained by adjusting the molar ratio of CHPTAC to anhydroglucose unit (AGU) of cellulose and the reaction time. The QC solutions in water displayed a typical polyelectrolyte behavior, and the intrinsic viscosity ([η]) value determined from the Fuoss-Strauss method increased with increasing DS value. Moreover, two QC samples (DS ) 0.46 and 0.63) were selected and studied as gene carriers. The results of gel retardation assay suggested that QCs could condense DNA efficiently. QCs displayed relatively lower cytotoxicity as compared with PEI, and QC/DNA complexes exhibited effective transfection compared to the naked DNA in 293T cells. The quaternized cellulose derivatives prepared in NaOH/urea aqueous solutions could be considered as promising nonviral gene carriers.

Introduction Cationic cellulose derivatives are large-scale commercial products, having many useful characteristics, such as hydrophilicity, biodegradability, and antibacterial properties.1–3 Hence, cationic cellulose derivatives have been found in numerous applications in a variety of fields, including the paper and textile, food, cosmetics, chemical, and pharmaceutical industries.4 The interactions, such as binding process, gelation properties, and phase behavior, of cationic cellulose derivatives with different surfactants have been extensively studied.2,5–10 The rheological properties have also been studied to evaluate the interactions between cationic celluloses and polyanions in water.1,11 The rheology of cationic cellulose ethers can be modulated by the addition of a small amount of an oppositely-charged polymer, leading the way to the design of novel pH-sensitive fluids.1 Moreover, cationic celluloses can be chemically cross-linked by ethylenglycol diglycidylether (EGDE) to prepare pH-/ionsensitive hydrogels, which have a wide range of potential applications in the biomedical and pharmaceutical fields.12–14 More recently, the physicochemical and transfection properties of cationic hydroxyethylcelluloses/plasmid DNA (pDNA) nanoparticles were investigated.3 Although the cationic cellulose/ pDNA nanoparticles tranfected cells to a much less extent than the polyethylenimine (PEI)-based pDNA nanoparticles, tailoring the nature and extent of cationic side chains on the cationic celluloses may be promising to further enhance their DNA delivery properties.3 Nowadays, commercial cationic cellulose derivatives are mainly the quaternization of water-soluble hydroxyethylcelluloses. The cationization of cellulose is mostly limited to the modification of pulp and fiber to improve their paper-making properties15,16 and dye uptakes,17,18 respectively. Pasˇteka reported the homogeneous quaternization of regenerated cellulose * To whom correspondence should be addressed. Tel.: +86-27-87219274. Fax: +86-27-68754067. E-mail: [email protected].

with 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) in solutions of benzyltriethylammonium hydroxide.19 However, the reported degree of substitution (DS) of the quaternized products was as low as 0.25. Heinze et al. have synthesized 6-deoxy-6-trialkylammonium cellulose derivatives from cellulose sulfonates.20 The resulting cationic derivatives were water-soluble even at low DS value, that is, in the range of 0.2-0.5. Until now, cationic cellulose derivatives prepared from cellulose directly by a homogeneously process have been scarcely reported20 because of the insolubility of cellulose in water and in most organic solvents on account of their strong inter- and intramolecular hydrogen bonding. In the present work, we reported the homogeneous quaternization of cellulose in an aqueous solution system for the first time. Cellulose was dissolved in NaOH/urea aqueous solutions directly to prepare a transparent solution, and then CHPTAC was used as etherifying agent reacted with cellulose under alkaline conditions. The structure and properties of the quaternized celluloses were studied. Moreover, cytotoxicity and transfect efficiency of the cationic cellulose/DNA complexes were investigated.

Experimental Section Materials. Cellulose (cotton linter pulp) was supplied by Hubei Chemical Fiber Group Ltd. (Xiangfan, China), and the viscosity-average molecular weight (Mη) of the cellulose was determined by viscometry in cadoxen21 to be 7.8 × 104. CHPTAC was purchased from Guofeng Fine Chemical Co. Ltd., Shandong, China, and was used as etherifying reagents without further purification. Branched polyethylenimine, with a molecular weight of 25 kDa, was purchased from Sigma-Aldrich. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT), and Dulbecco’s phosphatebuffered saline (PBS) were purchased from Invitrogen Corp. All other reagents were of analytical grade and were used without further purification. Quaternization of Cellulose. Cellulose solution was prepared according to the previous method.22 Into a 250 mL beaker, an adequate

10.1021/bm800429a CCC: $40.75  2008 American Chemical Society Published on Web 07/19/2008

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Table 1. Conditions and Results of the Homogeneous Quaternization of Cellulose with CHPTAC in NaOH/Urea Aqueous Solution sample ID molar ratioa time (h) temp (°C) nitrogen (%)

DS

QC-1 QC-2 QC-3 QC-4 QC-5 QC-6 QC-7 QC-8 QC-9 QC-10

0.20 0.29 0.31 0.46 0.47 0.63 0.35 0.46 0.42 0.44

a

3 4.5 6 9 12 12 9 9 9 9

8 8 8 8 8 16 4 16 8 8

25 25 25 25 25 25 25 25 45 60

1.453 1.975 2.058 2.777 2.809 3.420 2.258 2.773 2.609 2.705

The molar ratio of CHPTAC to AGU.

amount of NaOH, urea, and distilled water (7.5:11:81.5 by weight) were added, and the resulting aqueous solution was stored in a refrigerator. After the solution was precooled to -12.3 °C, cellulose was added immediately into it with vigorous stirring for 5 min at 25 °C to obtain the transparent cellulose dope (2 wt %). The cellulose solution was subjected to centrifugation at 8000 rpm for 20 min at 10 °C to exclude the slightly remaining undissolved part before using in further. In a typical reaction procedure, a certain amount of CHPTAC aqueous solution was added dropwise into the 100 g cellulose solution obtained previously, and the mixture was stirred at 25 °C. The reaction product was neutralized with aqueous HCl and dialyzed with regenerated cellulose tubes (Mw cutoff 8000, U.S.A.) against distilled water for 7 days. The solution was finally freeze-dried with lyophilizer (Christ Alpha 1-2, Osterode am Harz, Germany) to obtain the purified cellulose derivative (white powder). According to Table 1, 10 quaternized cellulose derivatives (coded as QCs) were prepared by changing the mole ratio of CHPTAC to the anhydroglucose unit (AGU) and the reaction conditions such as time and temperature. Characterization of QCs. FTIR spectra of QC samples and cellulose were performed with a Nicolet 170SX Fourier transform infrared spectrometer. The test specimens were prepared by the KBr-disk method. Nitrogen contents (N %) of QCs were measured with an elemental analyzer (CHN-O-Rapid, Foss Hera us GmbH, Hanau, Germany). The DS value of QC was determined by nitrogen content and calculated according to the following eq 1:

DS ) 162 × N % ⁄ (14 - 151.5 × N %)

(1)

C NMR measurements of the samples in D2O at 25 °C were carried out on a Varian INOVA-600 spectrometer in the proton noisedecoupling mode with a standard 5 mm probe at ambient temperature, and the sample concentration was about 3.5 wt %. The chemical shifts were referenced to the signals of tetramethylsilane (TMS). Solubility of the QCs in distilled water and in different solvents was measured at 25 °C, and the polymer concentration was about 1% (w/ v). ζ-Potential of the QCs in distilled water (cpolymer ) 1 mg/mL) was performed on a Nano-ZS ZEN3600 (Malven Instruments, U.K.) at 25 °C. The viscosity of the samples in pure water and in 0.1 mol/L NaCl aqueous solutions was measured at 25 ( 0.1 °C with an Ubbelodhe capillary viscometer. The kinetic energy correction was always negligible. Size exclusion chromatography (SEC) combined with laser light scattering (LLS) was used to determine the weight-average molecular weight (Mw) of QCs. The SEC-LLS measurement was performed on a multiangle LLS instrument (DAWN DSP, Wyatt Technology Co., U.S.) equipped with a He-Ne laser (λ ) 632.8 nm) and combined with a p100 pump (Thermo Separation Co. USA) equipped with TSK GEL G4000 PWXL column (7.8 mm × 300 mm) and an Optilab refractometer (Wyatt Technology) at 25 °C. The fluent was 0.15 M NaCl aqueous solution at a flow rate of 0.5 mL/min. Astra software was used for data acquisition and analysis. The specific refractive-index increments of QC in 0.15 M NaCl aqueous solution was determined 13

with an Optilab refractometer (Wyatt Technology) at 632.8 nm and 25 °C and was found to be 0.135 cm3/g. Gene Carrier Assay. Cell Culture. Human embryonic kidney transformed (293T) cells were incubated in DMEM containing 10% FBS and 1% antibiotics (penicillin-streptomycin, 10000 U/mL) at 37 °C in a humidified atmosphere containing 5% CO2. Amplification and Purification of Plasmids. In this study, pGL-3 plasmid used as the luciferase report gene was transformed in E. coli JM109. pGL-3 plasmid was amplified in terrific broth media at 37 °C overnight at 300 rpm. Then the purified plasmid was diluted by TE buffer solution and stored at -20 °C. The integrity of plasmid was confirmed by agarose gel electrophoresis. The purity and concentration of plasmid were determined by ultraviolet (UV) absorbance at 260 and 280 nm. Agarose Gel Electrophoresis Assay. Agarose gel electrophoresis experiments were performed to monitor the complexation of QC to DNA. Two series of QC/DNA complexes (QC-4 and QC-6) at different N/P ratios (the quaternary ammonium salt groups on cellulose backbone to phosphate groups of DNA) ranging from 0.5 to 6 were prepared by adding appropriate volume of QC (in 0.15 M NaCl solution) to 96 ng (0.8 µL) of pGL-3 DNA (120 ng/µL in 40 mM Tris-HCl buffer solution). The complexes were diluted by 0.15 M NaCl solution to 6 µL, and then the complexes were incubated at 37 °C for 30 min. After that, the complexes were subjected to electrophoresis on the 0.7% (W/ V) agarose gel containing GelRed and with Tris-acetate (TAE) running buffer at 80 V for 80 min. DNA was visualized with a UV lamp using a Vilber Lourmat imaging system. Particle Size and ζ-Potential Measurements. Particle size and ζ-potential of QC/DNA complexes were measured by Nano-ZS ZEN3600 (Malven Instruments, U.K.) at 25 °C. The complexes at various N/P ratios ranging from 1 to 30 were prepared by adding appropriate volume of QC (QC-4 and QC-6) solution (in 0.15 M NaCl solution) to 1 µg of pGL-3 DNA solution (in 40 mM Tris-HCl buffer solution). The complexes were incubated at 37 °C for 30 min. After that the complexes were diluted by 0.15 M NaCl solution to 1 mL prior to measurement. Cytotoxicity Assay. The cytotoxicity of QCs (QC-4 and QC-6) and 25 kDa PEI was examined by MTT assay. The 293T cells were seeded in a 96-well plate at 6000 cells/well and cultured for 1 day in 200 µL of DMEM containing 10% FBS. After the polymers were added for 2 days, the medium was replaced with 200 µL of fresh medium. Then 20 µL of MTT solutions were added for 4 h. After that, the medium was removed and 150 µL of DMSO was added and mixed. The absorbance was measured at 570 nm using a microplate reader (BIORAD, Model 550, U.S.A.). The relative cell viability was calculated as

cell viability (%) ) (OD570(samples) ⁄ OD570(control)) × 100 where OD570(control) was obtained in the absence of polymers and OD570(samples) was obtained in the presence of polymers. In Vitro Transfection (Luciferase Assay). For the in vitro transfection studies, the 25 kDa PEI/DNA at N/P ratio of 10 and naked pGL-3 DNA (1 µg) were respectively used as the positive and negative control. The 293T cells were seeded at a density of 6 × 104 cells/well in 24well plate with 1 mL DMEM containing 10% FBS and incubated at 37 °C for 24 h. The QC/DNA complexes at N/P ratios ranging from 5 to 30 were prepared by adding an appropriate volume of QC to 1 µg of pGL-3 DNA solution and then diluted by 0.15 M NaCl solution to 100 µL and incubated at 37 °C for 30 min. Before transfection, the cells were washed by PBS, and then the QC/DNA complexes, PEI/ DNA complexes, and naked DNA were added with serum-free DMEM for 4 h at 37 °C. After that the serum-free DMEM was replaced by fresh DMEM containing 10% FBS, and the cells were further incubated for 2 days. The luciferase assay was performed according to manufacture’s protocols. Relative light units (RLUs) were measured with chemiluminometer (Lumat LB9507, EG&G Berthold, Germany). The total protein was measured according to a BCA protein assay kit (Pierce). Luciferase activity was expressed as RLU/mg protein. Data

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Scheme 1. Homogeneous Quaternization of Cellulose with 3-Chloro-2-hydroxypropyl-trimethylammonium Chloride (CHPTAC) in NaOH/Urea Aqueous Solutions

Figure 1. FTIR spectra of the native cellulose and the quaternized cellulose sample (QC-4).

were shown as mean ( standard deviation (SD) based on three independent measurements.

Results and Discussion Homogeneous Synthesis of Quaternized Celluloses. Scheme 1 illustrates the homogeneous quaternization of cellulose dissolved in NaOH/urea aqueous solution by using CHPTAC as etherifying agent. Under the alkaline conditions, epoxide is produced in situ from CHPTAC, and quaternized cellulose is then formed through reaction between the cellulose sodium alkoxide and the epoxide or CHPTAC. The main reaction is the cationization reaction of cellulose, but also some diols are formed as a result of the side reaction.23–25 The reaction conditions for quaternization of cellulose are summarized in Table 1. During the process of quaternization, the solution kept its transparent nature and remained completely homogeneous as the reaction proceeded. The content of nitrogen and the degree of quaternization (expressed as DS value) under various reaction conditions are shown in Table 1. The N content and DS value of the quaternized derivatives increased with increasing molar ratio of CHPTAC to AGU, as well as the reaction time. However, the reactions were hardly affected by the reaction temperature because the products displayed almost similar N content and DS value as the reaction temperature increased from 25 to 65 °C. Further increase in temperature could result in the gelation of cellulose solution. Therefore, water-soluble quaternized cellulose derivatives with the DS values of 0.2 to 0.63 could be obtained by changing the molar ratio of CHPTAC/ AGU from 3 to 12 and the reaction time from 4 to 16 h. For the first time, we synthesized homogeneously the quaternized cellulose derivatives in aqueous solutions without addition of an extra base as a result of the basic nature of our solvent system. Structure Analysis. Figure 1 shows the FTIR spectra of the native cellulose and the quaternized cellulose samples. The native cellulose showed a broadband at about 3400 cm-1, assigned to stretching vibration modes of O-H groups. The bands at 2900, 1375, and 1062 cm-1 were assigned to stretching vibration of -CH2- groups. The most striking difference between native cellulose and QCs spectra was the peak obtained for QC at 1482 cm-1, which corresponded to the methyl groups of ammonium.26 Moreover, the peak of QCs positioned at 1414 cm-1 was referenced as the C-N stretching vibration.27,28 FTIR spectra have given an evidence of the introduction of the quaternary ammonium salt group on the cellulose backbone. The 13C NMR spectra of QC-3 (DS ) 0.31) and QC-6 (DS ) 0.63) in D2O at 25 °C are shown in Figure 2. The peaks

Figure 2. 13C NMR spectra of the quaternized cellulose samples (a, DS ) 0.31; b, DS ) 0.63) in D2O at 25 °C.

were assigned according to the quaternary ammonium derivatives of konjac glucomannan25 and starch.23,24 The peak at the lower field (102.8 ppm) corresponded to Cl and C1′ (the peak for C1 was influenced by the reaction at O-2); the peak at 60.2 ppm was assigned to the pendant methylene carbon C6. The primary hydrogel groups bearing cationic functions gave a new signal at around 68.4 ppm, which was overlapped by the chemical shift of C9. Signals at 73.4, 65.4, and 68.4 ppm were assigned for the C7, C8, and C9 atoms, respectively. The typical signal of the (CH3)3N+ moiety appeared at 54.5 ppm. The rest of carbon atoms present in the basic polymer backbone were determined between 70 and 85 ppm (Figure 2). Therefore, the results of NMR further proved the successful synthesis of quaternized celluloses in NaOH/urea aqueous solutions. Solution Properties. Table 2 lists the solubility of the quaternized cellulose derivatives in different aqueous solutions. All of the 10 QC samples exhibited good solubility in pure water. QCs with DS value higher than 0.35 showed good solubility in 0.1 M NaCl and HCl aqueous solutions. However, QCs with DS value higher than 0.47 displayed good solubility

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Table 2. Solubility, Intrinsic Viscosity, and ζ-Potential of QCs solubilitya

[η] (mL/g)

sample ID

H2O

0.1 M NaCl

0.1 M HCl

0.1 M NaOH

QC-1 QC-2 QC-3 QC-4 QC-5 QC-6 QC-7 QC-8 QC-9 QC-10

++ ++ ++ ++ ++ ++ ++ ++ ++ ++

∆ + + ++ ++ ++ ++ ++ ++ ++

∆ + + ++ ++ ++ ++ ++ ++ ++

∆ ∆ ∆ + ++ ++ ∆ + + +

a

H2O 3616 4828 5049 8064 10219 11172 7158 10452 9173 10775

0.1 M NaCl

ζb (mV)

332 300 295 337 362 334 332

25.6 26.8 31.6 43.6 43.0 44.6 33.7 43.2 33.1 35.6

+ soluble, ++ good solubility, and ∆ swelling for a 1% (w/v) solution at 25 °C. b ζ-Potential determined by the 1 mg/mL aqueous solutions at 25 °C.

Figure 3. Dependence of the reduced viscosity (ηsp/c) on concentration (c) for the QC-6 sample in pure water at 25 °C. Inset is the dependence of c/ηsp on c1/2.

Figure 4. SEC chromatograms of the quaternized celluloses in 0.15 M NaCl aqueous solutions at 25 °C, with a flow rate of 0.5 mL/min.

in 0.1 M NaOH aqueous. The relatively lower limit of DS values for water-soluble QCs in this work may be due to the homogeneous substituent distribution within an AGU unit and along the molecular chain. The ζ-potential values for QCs in pure water are listed in Table 2. An increase in ζ-potential value from 25.6 to 44.6 mV with increasing of DS value from 0.2 to 0.63 has been observed. The reduced viscosity ηsp/c of QC-6 in pure water at 25 °C is shown in Figure 3. In the absence of salt, the reduced viscosity of QC displays a typical electrostatic repulsion effect, which remarkably increases with a decrease of the polymer concentration, as a result of the expanded polyelectrolyte chains and chain-chain repulsion.29 Intrinsic viscosity ([η]) is a measure of hydrodynamic volume of macromolecules. It is generally accepted that macromolecule conformation and molecular weight play a fundamental role, through their relationships with the molecular dimensions and shapes, in determining the value of [η]. Therefore, the [η] values could reflect the expanded extent of the polymer chain.30,31 Fuoss and Strauss32 had shown that (ηsp/c) versus c plots for polyelectrolytes are strongly concaved upward, and then they proposed an empirical expression to estimate the intrinsic viscosity ([η]), as follows in eq 2

increased for the more extended chain conformation, which attributed to the electrostatic repulsion of the polyelectrolyte solutions. The electrostatic interactions have been screened with the addition of NaCl, leading to the normally viscosity behavior of QC solutions like to neutral polymers. So the solution viscosity decreased gradually with the addition of NaCl. As shown in Table 2, the [η] values of QCs in 0.1 M NaCl aqueous solutions are lower than that obtained in water. Moreover, the [η] values of QC-5 and QC-6 in 0.1 M NaCl aqueous solutions are lower than those of the other QC samples, owing to the aggregation of QCs in solution. SEC chromatograms of the QCs in 0.15 M NaCl aqueous solutions by SEC-LLS at 25 °C are shown in Figure 4. The chromatograms of QC-6 (DS ) 0.63) and QC-5 (DS ) 0.47) displayed a signal peak, and the corresponding weight-average molecular weight (Mw) was determined to be 9.30 × 104 and 9.85 × 104, respectively. The Mw of QC-6 and QC-5 are in good agreement with the molecular weight calculated from the DS value and the Mη of the native cellulose (7.8 × 104). This suggests that NaOH/urea aqueous solution is a stable system for the cellulose derivatization. When the DS value of QC was lower than 0.47, the chromatograms obtained both by LLS and by the refractive index detectors exhibited two peaks, indicating the occurrence of aggregation by the biomodal distribution. With decreasing of DS value, the aggregation of QC became stronger, which attributed to the strong hydrogen bonding of the residual -OH groups within the AGU unit of the derivatives. Interactions of QCs with pDNA. The binding capability of polycations to pDNA is a prerequisite as the gene vectors. The polycations can condense pDNA into compact structures and

ηsp ⁄ c ) [η] ⁄ (1 + Bc1⁄2)

(2)

where B is a constant to account for the interactions of polyelectrolytes. By plotting (c/ηsp) versus c1/2, a linear relationship (as shown in the inset of Figure 3) can be found with an intercept of 1/[η], and slope of B/[η]. The [η] values of QCs in pure water obtained by fitting the Fuoss equations are listed in Table 2. With an increasing DS value of QCs, the [η] value

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Figure 5. Agarose gel electrophoresis retardation assay of (a) QC4/DNA complexes and (b) QC-6/DNA complexes. Plasmid DNA (96 ng) was mixed with QCs at different N/P ratios: 1(0), 2(0.5), 3(1), 4(2), 5(3), 6(4), 7(5), 8(6).

reduces the electrostatic repulsion between DNA and cell surface by neutralizing the negative charge, which facilitates the uptake of the complex to negatively charged cell membrane constituents and, therefore, to a higher rate of uptake. When pDNA is condensed by polycations, it also can be better protected against enzymatic degradation by nucleases in serum and extracellular fluids.33 In this work, the binding capability of QC-4 and QC-6 to pDNA was studied by agarose gel electrophoresis, and plasmid DNA (96 ng) was mixed with QC samples at different N/P ratios. The gel electrophoresis measurements in Figure 5 clearly show that, at N/P ratio of 2 and higher, both QC-4 and QC-6 bound the pDNA completely. Moreover, the binding ability of QC-6 to pDNA was stronger than that of QC-4 on account of its relatively higher DS value, which induced higher density of positive charge. The charge and particle size of the polymer/DNA complexes are very important for polycations used as gene vectors34 and play an important role in the entering of complex into nucleolus by cellular uptake.33 In this study, the particle sizes and ζ-potential of QC-4/DNA and QC-6/DNA complexes at various N/P ratios ranging from 1 to 30 were measured by adding QC solution to 1 µg DNA at physiological ionic strength condition (in 0.15 M NaCl solution). As shown in Figure 6a, the particle size of QC-4/DNA and QC-6/DNA complexes at a N/P ratio of 1 were 1380 and 1070 nm, respectively. As the N/P ratios increased to 2, the particle size of QC/DNA complexes decreased sharply as a result of the net positive electrostatic repulsion between complexes. The particle size of QC-4/DNA and QC-6/DNA complexes have the similar trends (between 390 and 550 nm) at various N/P ratios ranging from 2 to 30. Figure 6b showed the ζ-potential values of QC/DNA complexes at N/P ratios ranging from 1 to 30. For QC-4/DNA complexes, the ζ-potential values increased from -7.0 to 25.1 mV with increasing of N/P ratios from 1 to 3 and then hardly increased with further increasing of N/P ratios. Similarly, the ζ-potential values of QC-6/DNA complexes increased from -0.9 to 31.1 mV as the N/P ratios increased from 1 to 5 and then hardly changed with further increasing of N/P ratios. At the same N/P ratios, the ζ-potential values of QC-6/DNA complexes were larger than those of QC-4/DNA complexes, which attributed to the relative higher DS value and larger ζ-potential value of QC-6 than those of QC-4 (as shown in Tables 1 and 2). In Vitro Cytotoxicity and Transfection. One of the major requirements of the polymeric vectors for use in gene therapy is the absence of cytotoxicity. In our work, the cytotoxicity of QC-4 and QC-6 was evaluated in 293T cells by MTT assay, and the 25 kDa PEI was used as the control. As shown in Figure 7, the cytotoxicity of QCs increased gradually with increasing of the concentrations and DS values due to the presence of a higher amount of cationic cellulose damaging the cellular membranes. However, the QCs exhibited much lower cytotox-

Figure 6. (a) Particle size and (b) ζ-potential of QC/DNA complexes at N/P ratios ranging from 1 to 30. Data are shown as mean ( SD (n ) 3).

Figure 7. Cell viabilities of 293T cells in the presence of QC-4, QC6, and 25 kDa PEI. Data are shown as mean ( SD (n ) 3).

icity when compared with PEI, the most popular “gold standard” of cationic polymer transfection.35 Anyhow, the fact that QCs seemed to be well tolerated by cells was encouraging for further transfection experiments. The transfection efficiency of QC/DNA complexes was evaluated in 293T cells at N/P ratios ranging from 5 to 30, using naked DNA and 25 kDa PEI/DNA complexes at its optimal ratio (N/P ) 10) as the controls. The plasmid pGL-3 DNA was used as the luciferase reporter gene. On the basis of the results

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Acknowledgment. This work was financially supported by the National High Technology Research and Development Program of China (2004AA649250) and the National Natural Science Foundation of China (20204011, 20674057, and 30530850).

References and Notes

Figure 8. Luciferase expression in 293T cells transfected by QC/ DNA complexes at different N/P ratios. Transfection efficiency of the naked DNA and 25 kDa PEI/DNA complexes at N/P ratio of 10 are shown as control. Data are shown as mean ( SD (n ) 3).

of cytotoxicity (Figure 7) at the N/P ratio of 30, the viability of 293T cells is above 90% at the concentration of 46 µg/mL for QC-4/DNA complex and 37 µg/mL for QC-6/DNA complex, respectively. Hence, the N/P ratios ranging from 5 to 30 are fit for the transfection experiments. Figure 8 showed the transfection efficiency of QC/DNA complexes, PEI/DNA complexes, and naked DNA. It indicated that the trends of transfection efficiency for QC-4/DNA and QC-6/DNA complexes were similar, and the transfection efficiency depended on the DS value of QCs. Clearly, the transfection efficiency of QC-6/DNA complexes at various N/P ratios was higher than that of QC4/DNA complexes. The relatively higher transfection efficiency of the QC-6/DNA complexes was attributed to the positively charged surface, which allows an optimal binding of QC-6/DNA complexes to cellular membranes. As shown in Figure 8, although the transfection efficiency was relatively lower than that of PEI, QCs increased the transfection efficiency markedly compared with the naked DNA values (up to 1058-fold). Considering the much lower cytotoxicity compared with PEI, QCs could be considered as promising nonviral gene carriers in vitro.

Conclusions Cationic derivatives of cellulose were homogeneously synthesized, for the first time, by reacting cellulose with CHPTAC in NaOH/urea aqueous solutions. Water-soluble QCs with DS value of 0.20-0.63 could be obtained by adjusting the molar ratio of CHPTAC to AGU of cellulose from 3 to 12, and the reaction time from 4 to 16 h. Aqueous solutions of QCs displayed a typical polyelectrolyte behavior, and the [η] value calculated from the Fuoss-Strauss method increased with increasing DS value, indicating a more extend chain conformation. QCs could condense DNA efficiently. The cytotoxicity of QCs was evaluated in 293T cells and was found to be relatively low compared with PEI. The transfection efficiency of the QC/ DNA complexes was measured by the luciferase gene in 293T cells, and the cells had been transfected effectively. Both the cytotoxicity of QCs and the transfection efficiency of QC/DNA complexes increased with increasing DS of QC. The results indicated that the quaternized cellulose derivatives obtained in the aqueous system are promising agents to be used as gene carriers.

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