Influence of Disulfide Density and Molecular Weight on Disulfide

Feb 3, 2009 - Phone: +86-27-6875-4061. ... The cross-linked PEI800-SSY polymers with a moderate thiolation ... A suitable molecular weight of raw PEI ...
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Bioconjugate Chem. 2009, 20, 340–346

Influence of Disulfide Density and Molecular Weight on Disulfide Cross-Linked Polyethylenimine as Gene Vectors Qi Peng, Chu Hu, Juan Cheng, Zhenlin Zhong,* and Renxi Zhuo Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China. Received October 20, 2008; Revised Manuscript Received December 30, 2008

Disulfide cross-linked polyethylenimines (PEIX-SSY, where X refers to the molecular weight of raw PEI, and Y refers to the thiolation degree) were prepared in two steps: First, thiol groups were introduced on a raw polyethylenimine (PEI) by the amine-induced ring-opening reaction of thiirane. Second, thiol groups were oxidized by DMSO to form the disulfide cross-links. The cross-linked PEI800-SSY polymers with a moderate thiolation degree (PEI800-SS2.6, PEI800-SS3.5, and PEI800-SS4.5) could form compact polyplexes with a size of 200-300 nm at an adequate N/P ratio. In contrast, those with a too low or too high thiolation degree (Y below 2.6 or above 4.5) formed much looser polyplexes with a size above 600 nm. The polyplexes of PEIX-SS3.0-4.0 series (X ) 800, 1800, and 25,000) formed small particles with a size below 400 nm at a wide range of N/P ratios. Efficiency of the cross-linked PEIs as gene vectors was evaluated in vitro by transfection of pGL3 to HeLa, COS7, 293T, and CHO cells. The efficiency is disulfide content and molecular weight dependent. The PEI800-SSY series with an adequate thiolation degree between 2.6 and 4.5 have relatively lower cytotoxicity and higher gene transfection efficiency than 25 KDa PEI. The polymers with very low or very high thiolation degrees were unable to form compact polyplexes and had very poor transfection efficiency. A suitable molecular weight of raw PEI is also essential to obtain a highly efficient disulfide cross-linked PEI gene vector. Among the three raw PEIs of different molecular weights tested (800 Da, 1800 Da, and 25 KDa), the cross-linked polymer prepared from 800 Da PEI that has the lowest molecular weight gave the best results.

INTRODUCTION Polymer-mediated gene delivery has recently appeared as a comparable alternative to viral vectors for its lower cost, large nucleic acid loading capacity, and no immunogenesis (1-3). For example, polyethylenimine (PEI) is one of the most effective nonviral gene delivery carriers. The proton sponge nature of PEI is thought to cause osmotic swelling and physical rupture of the endosome, resulting in the escape of the vector from the degradative lysosomal trafficking pathway (4, 5). High molecular weight is necessary for effective gene transfection with PEI. While high molecular weight PEI possesses high gene transfection efficiency, it has a high cytotoxicity (6, 7). Comparatively, low molecular weight PEI showing poor gene transfection efficiency was reported to have much lower cytotoxicity (8). Therefore, a reasonable way for resolving the contradiction between efficiency and cytotoxicity was suggested to combine low molecular weight PEIs together with degradable links such as amides, acetals, esters, and disulfides. It was expected to produce a polymer with lower toxicity and relatively high gene transfection efficiency, as well as long-term safety. Recently, many kinds of degradable PEIs showed good transfection efficiency and low cytotoxicity (9-12). A disulfide bond can be broken in the presence of glutathione (GSH). The intracellular glutathione concentration is 50-1000 times higher than the extracellular concentration (13-15). The presence of disulfide in gene vectors could maintain a balance of keeping pDNA polyplexes stable in the extra-cytoplasmic environment and inducing efficient release of the entrapped pDNA from the polyplexes after their movement into the reductive cytoplasmic compartment. It also would limit the * To whom correspondence and reprint requests should be addressed. Phone: +86-27-6875-4061. Fax: +86-27-6875-4509. E-mail: [email protected].

toxicity of the polymers. The advantages of disulfide linkages in gene transfection polymers are exemplified in the literature (11, 16-20). Bioreducible polyamidoamines containing multiple disulfide linkages with low toxicity were able to transfect COS7 cells in vitro with transfection efficiencies significantly higher than those of 25 kDa PEI (17). Linear poly(ethylenimine sulfide)s were reported to be safe gene carriers with high transfection efficiencies comparable to 25 kDa PEI (19). Branched PEI with an Mw of 800 Da was cross-linked by the reaction with either dithiobis(succinimidylpropionate) (DSP) or dimethyl 3,3′-dithiobispropionimidate dihydrochloride (DTBP), resulting in greatly increased gene transfection efficiency. Also, cytotoxicity was decreased by avoiding long-term polymer accumulation (21). Recently, we demonstrated that disulfide cross-linked PEIs synthesized by oxidation of thiolated 800 Da PEI possess simultaneously higher gene transfection efficiency and lower cytotoxicity than 25 KDa PEI (22). It is reasonable to assume that the properties of disulfide crosslinked PEIs as gene vectors would be determined by (i) the type and density of disulfide cross-linkages and (ii) the molecular weights of both raw and cross-linked PEIs. In this paper, the influence of disulfide density and molecular weights of raw PEIs on the molecular weights and transfection efficiency of the crosslinked PEIs is investigated.

MATERIALS AND METHODS Materials. PEIs with Mw of 800 Da and 25 kDa were purchased from Aldrich, and PEI with a Mw of 1800 Da was from Alfa Aesar. Thiirane was synthesized by literature procedure (23). Plasmid pGL3 under the control of SV40 promoter and with enhancer sequences encoding luciferase was obtained from Promega, Madison, WI, USA. Plasmids were propagated in Escherichia coli in Luria-Bertani (LB) medium containing 60 µg/mL ampicillin respectively at 37 °C and

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Table 1. Synthesis of PEIX-SSYa starting materials PEIX-SSY PEI800-SS0.4 PEI800-SS1.0 PEI800-SS2.6 PEI800-SS3.5 PEI800-SS4.5 PEI800-SS5.5 PEI800-SS7.0 PEI1800-SS3.5 PEI25K-SS4.0

results

SH content wt of raw PEI thiirane thiirane/PEI in PEIX-SHY PEIX-SSY (Wt/g) (Wt/g) (molar ratio) (SH/PEI) (g) 1.436 1.314 0.667 1.026 0.990 0.536 0.730 1.088 3.878

0.051 0.120 0.200 0.375 0.458 0.300 0.45 0.132 0.039

0.47 1.22 4.00 4.9 6.17 7.46 8.22 3.64 4.19

0.4 1.0 2.6 3.5 4.5 5.5 7.0 3.5 4.0

1.040 1.177 0.875 1.548 1.664 0.980 1.383 1.224 5.894

a Reaction conditions: Step 1 (thiolation): pH 7.2 in CH3OH, 50 °C for 24 h. Step 2 (oxidation): room temperature in DMSO for 2 days.

purified using E. Z. N. A. Fastfilter Endofree Plasmid Midi kits (Omega) according to the manufacturer’s instruction. The purity of DNA was assessed spectrophotometrically by measuring absorbance at wavelengths of 260 and 280 nm (OD260/OD280 1.8 or greater) and confirmed using 0.7% agarose gel electrophoresis containing GelRed. The DNA concentration was determined by measuring the UV absorbance at 260 nm. DNA aliquots of pGL3 were stored at -20 °C prior to use. Dulbecco’s modified eagle’s medium (DMEM), penicillin-streptomycin, trypsin, Dulbecco’s phosphate buffered saline, and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma. Other commercially available reagents were used as received. Synthesis of Thiolated PEI (PEIX-SHY). In a 25 mL flask, 1.00 g of PEI (800 Da, 1800 Da, or 25 kDa) was dissolved in 5 mL of deionized water. Hydrochloric acid (0.1 N) was added dropwise to the PEI solution while stirring until pH was regulated to 7.2. After removal of water by evaporation under reduced pressure, the yellow solid thus obtained was dissolved in 3 mL of CH3OH and transferred to an ampule. After the container was purged with argon for 5 min, a calculated amount of thiirane was added. The ampule was sealed and kept in a 50 °C oil bath for 24 h with magnetic stirring. The content of the ampule was transferred to a flask, evaporated to dryness under reduced pressure, and stored under argon. The thiol group content was determined by Ellman’s method (24, 25) and listed in Table 1. 1H NMR (300 MHz, D2O): δ 3.15-2.55 (m, NCH2CH2N, NCH2CH2S). Synthesis of Disulfide Cross-Linked PEI (PEIX-SSY). A solution of 1.50 g of PEIX-SHY in 3 mL of methanol and 1.5 mL of dimethyl sulfoxide (DMSO) was stirred for 48 h. The reaction solution was precipitated in diethyl ether three times and dried under high vacuum to give a light yellow viscous glue or solid. 1H NMR (300 MHz, D2O): δ 3.14-2.66 (m, NCH2CH2N, NCH2CH2S). Cytotoxicity Assay of PEIX-SSY. The cytotoxicity assay of PEIX-SSY was performed the same way as described in the previous paper (17). Molecular Weight Determination. Capillary viscosity measurements were carried out to estimate molecular weights (26, 27). The polyamine polymers were neutralized with hydrochloric acid to pH 7.4 and dissolved in 150 mM NaCl at concentration from 20 mg/mL to 5 mg/mL. Viscosity measurements were carried out using a capillary viscometer at 25 °C. The molecular weights of cross-linked polymers were calculated using the Mark-Houwink equation [η] ) KMR, where M is the molecular weight and K and R are Mark-Houwink parameters determined from PEI standards of known molecular weights. The molecular weights of the polymers were also determined by gel permeation chromatographic (GPC) using a Waters 2690D HPLC equipped with Ultrahydrogel 120 and 250

columns. HAc/NaAc (pH 2.8) buffer solution was used as the eluent at a flow rate of 0.3 mL/min. Poly(ethylene glycol) standards with narrow distribution were used to generate a calibration curve. Preparation of PEIX-SSY /pDNA Polyplexes and Agarose Gel Retardation. A 0.1 mg/mL aqueous solution of plasmid DNA (pGL3) was diluted to designed concentration (usually 20 µg/mL) using 150 mM NaCl solution under vortexing. Designed amounts of PEIX-SSY aqueous solutions were added slowly to the pDNA solutions. The amount of polymer added was calculated on the basis of chosen N/P ratios of PEIX-SSY/DNA (nitrogen atoms of the polymer over phosphates of pDNA). The mixture was incubated at room temperature for 30 min for the complex formation. A 5 µL sample of the polyplex solution mixed with 1 µL of 6 × A loading buffer was loaded to agarose gel. The polyplexes were electrophoresed on a 0.7% agarose gel containing GelRed with Tris-acetate (TAE) running buffer (pH 8) at 80 V for 80 min. DNA bands were visualized by an UV (254 nm) illuminator and photographed with a Vilber Lourmat imaging system (France). Particle Sizes and Zeta Potential Measurements. The particle size and zeta potential were measured with a Malvern Nano-ZS ZEN3600 Zeta-Sizer at room temperature. The polyplexes at various N/P ratios were prepared the same way as above. Then, the polyplexes were incubated at room temperature for 30 min. After that, the polyplexes were diluted by 150 or 10 mM NaCl solution to 1 mL volume prior to measure particle sizes or zeta potentials. Determination of in Vitro Transfection Efficiency. The in vitro transfection efficiency of PEIX-SSY/pGL3 polyplexes was evaluated in HeLa, COS7, 293T, and CHO cells by the procedure as described in the previous paper (17).

RESULTS AND DISCUSSION Preparation of PEIX-SSY. The strategy for preparation of PEIX-SSY is shown in Scheme 1 similar to that in our previous report (17) except using thiirane as a thiolation regent instead of methylthiirane. First, a raw PEI is thiolated by a ring-opening reaction with a relevant quantity of thiirane to give the thiolated product PEIX-SHY, where X refers to the molecular weight of the raw PEI and Y stands for the average number of thiol groups on each PEI molecule. Second, PEIX-SHY is cross-linked into a higher molecular weight polymer PEIX-SSY by disulfide-forming oxidation of the thiol groups with DMSO. Three kinds of PEIs with weight-averaged molecular weights (Mw) of 800 Da, 1800 Da, and 25 kDa, respectively, were thiolated through the ring-opening reaction of thiirane with the primary and secondary amino groups of PEI at pH 7.2 in methanol, affording the thiolated products PEIX-SHY. The thiolation degree Y was determined by Ellman’s test (24, 25) and listed in Table 1. As shown in the table, the thiolation degree was correlated to the feed ratio of thiirane/PEI and could be adjusted over a wide range. Thiolated PEIs were cross-linked by oxidation with DMSO at room temperature, and the final products PEIX-SSY were subjected to Ellman’s test for the estimation of residual thiol groups. After 2 days of oxidative cross-linking reaction in DMSO, the amount of remained thiol groups was found to be less than 10% of the initial value for all samples, indicating that the disulfide-forming oxidation was near completion. For PEI800-SS0.4, PEI800-SS1.0, PEI800-SS2.6, PEI800-SS3.5, PEI800SS4.5, and PEI1800-SS3.5, the reaction mixtures were clear solutions during the whole oxidation process. For PEI800-SS5.5, PEI800-SS7.0, and PEI25K-SS4.0, however, gelatinous products were obtained. The gels dissolved again to become solutions gradually in one to two days at room temperature. The dissolution of the

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Scheme 1. Preparation of Disulfide Cross-Linked Polyethylenimine PEIX-SSY (X and Y Refer to the Molecular Weight of Raw PEI and Thiolation Degree, Respectively)

Table 2. Cytotoxicity of PEIX-SSY, and the Size and Zeta Potential of PEIX-SSY/pDNA Polyplexes particle size (nm) polymers PEI800-SS0.4 PEI800-SS1.0 PEI800-SS2.6 PEI800-SS3.5 PEI800-SS4.5 PEI800-SS5.5 PEI800-SS7.0 PEI1800-SS3.5 PEI25K-SS4.0 800 Da PEI 25 KDa PEI

IC50

a

(µg/mL)

>1000 >500 63 50 41 35 34 25 7 >1000 7

b

zeta potential (mV)

c

N/P ) 10

N/P ) 20

N/P ) 30

N/P ) 40

N/P ) 10

N/P ) 20

N/P ) 30

N/P ) 40

1069 1153 460 380 891 1626 1196 294 225 1190 220

796 950 376 232 281 1051 680 397 236 1630 159

696 904 290 277 243 861 654 331 351 968 164

593 780 300 212 238 577 647 433 346 1038 149

13.8 17.1 13.6 18.4 19.1 20.1 21.0 15.2 14.7 5.8 35.4

14.7 20.7 15.1 20.9 21.1 23.7 23.5 15.8 16.0 11.6 35.2

14.3 23.2 22.8 19.6 25.8 23.9 23.4 13.5 16.1 9.8 31.6

15.4 21.8 25.3 24.5 30.3 23.3 27.7 13.9 15.9 10.7 32.3

a IC50 values measured on HeLa cells at 24 h after the addition of PEIX-SSY series by MTT assay. b Average particle sizes of PEIX-SSY/pGL3 measured by DLS at different N/P ratios in 150 mM NaCl. c Zeta potentials of PEIX-SSY/pGL3 measured at different N/P ratios in 10 mM NaCl.

gels could be explained by the transform of intermolecular disulfide bonds into intramolecular ones (17). Molecular weights of the PEI800-SSY series were estimated by viscosity measurements using a capillary viscometer. In 150

Figure 1. GPC traces of PEI800-SSY series, 800 Da and 25 kDa PEI in HAc/NaAc (pH 2.8) buffer solution.

mM NaCl aqueous solution (pH 7.4, 20 °C), the molecular weights of PEI800-SS0.4, PEI800-SS1.0, PEI800-SS2.6, PEI800-SS3.5, and PEI800-SS4.5 were estimated to be 1800, 2900, 4000, 8500, and 8700, respectively. The molecular weight increases with the increase of thiolation degree Y when Y is below 3.5. The disulfide cross-linked PEIs have very similar molecular weights regardless of their thiolation degree in the range from 3.5 to 4.5. This result suggests that the majority of disulfide crosslinks are intramolecular when the thiolation degree is high. Because of the phenomenon of partial gelation, we were unable to estimate the molecular weight of PEI800-SS5.5 and PEI800SS7.0. GPC analysis results (Figure 1) revealed that the introduction of disulfides for raw PEIs elevate the average molecular weights of the final products, and with the increase of the content of disulfide in the polymers, the molecular weights of the PEI800SSY (Y ) 0.4, 1.0, 2.6, and 3.5) correspondingly increased. All PEI800-SSYs had a similar peak nearly repeating with 800 Da PEIs. Maybe it can be explained that quite a lot of raw materials interfused the final products without reacting with thiirane. Compared to the raw material 800 Da PEI, PEI800-SS0.4 and PEI800-SS1.0 showed little improvement in their molecular weights. The peak shape of PEI800-SS0.4 seems almost the same as that of its raw materials. From PEI800-SS1.0, the cross-linked polymer was formed showing higher molecular weight from

Disulfide Cross-Linked Polyethylenimine

Bioconjugate Chem., Vol. 20, No. 2, 2009 343

Figure 2. The effect of increasing concentration of PEIX-SSY on the electrophoretic migration of pGL3 through an agarose gel. The polyplex composition (N/P ratio) was expressed as PEI nitrogen atoms per DNA phosphate. Plates a-g: PEI800-SS0.4, PEI800-SS1.0, PEI800-SS2.6, PEI800-SS3.5, PEI800-SS4.5, PEI800-SS5.5, and PEI800-SS7.0, respectively. Plates h and i: PEI1800-SS3.5 and PEI25K-SS4.0, respectively. Plate j: 800 Da PEI. Plate k: 25 KDa PEI.

the shorter outflow times of the apexes near the peak. The shapes of apexes of PEI800-SS3.5, PEI800-SS4.5, and PEI800-SS5.5 were nearly the same, and their molecular weights remained constant from the results. In HAc/NaAc buffer solution (pH 2.8, 40 °C), the molecular weights of PEI800-SS0.4, PEI800-SS1.0, PEI800-SS2.6, PEI800-SS3.5, PEI800-SS4.5 were estimated to be 1238, 1365, 1742, 2075, and 2192, respectively. The trend of the results was similar to that obtained from viscosity measurement. Cytotoxicity of PEIX-SSY. MTT assay was carried out to determine the cytotoxicity of the PEIX-SSY series with the IC50 values listed in Table 2. The PEI800-SSY series, regardless of the thiol group content, had a lower cytotoxicity compared to 25 kDa PEI in HeLa cells. PEI800-SS0.4 and PEI800-SS1.0 showed low cytotoxicity similar to 800 Da PEI. With the increasing content of disulfide in the polymers, their toxicity increased and reached a maximum of around 34 µg/mL (IC50) at high thiolation degrees of 5.5 and 7.0. As shown in Table 2, with the similar disulfide content, PEIX-SSY prepared from higher molecular weight raw PEI had higher cytotoxicity than that prepared from lower molecular weight raw PEI. PEI25K-SS4.0 showed toxicity similar to that of 25 kDa PEI.

In addition, PEIX-SSY might degrade in vivo into individual low molecular weight PEI molecules with relatively low toxicity, and those low molecular weight fragments of polymers would be cleared more easily from the body. Formation and Characterization of PEIX-SSY /pDNA Polyplexes. To mediate endocytosis through cell membrane, cationic polymers need to condense DNA into compact particles via electrostatic interactions between the positively charged polymers and the negatively charged phosphates on DNA backbones (28, 29). The polyplex of a disulfide cross-linked PEI with a pDNA was prepared by adding PEIX-SSY aqueous solution slowly to the pDNA solution. The amount of polymer added was calculated on the basis of chosen N/P ratios, nitrogen atoms of the polymer over phosphates of pDNA, in PEIX-SSY/ pDNA. Formation and characterization of the polyplexes was investigated by agarose gel retardation, particle size, and zeta potential. Gel retardation assay verified that PEIX-SSY series can condense plasmid DNA at low N/P ratios. As shown in Figure 2, as the concentration of the polymer increased, the capability of integrating with plasmid DNA was enhanced. 800 Da PEI formed stable complexes with DNA only at N/P ratios higher

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Figure 3. In vitro transfection efficiency of the polyplexes of pGL3 with PEI800-SSY (A) and PEIX-SS3.0-4.0 (B) series in HeLa, COS7, 293T, and CHO cells in comparison with 800 Da and 25 kDa PEI.

than 40. In contrast, 25 kDa PEI was the most efficient that it could completely retard DNA migration at a N/P ratio of 0.6. The required N/P ratios were 7.3, 6.3, 3.8, 2.5, 2.5, 2.5, and 2.5 for PEI800-SS0.4, PEI800-SS1.0, PEI800-SS2.6, PEI800-SS3.5, PEI800-SS4.5, PEI800-SS5.0, and PEI800-SS7.0 to completely retard the pDNA, respectively. The pDNA-binding capacity of PEI800SSY is greatly enhanced by the disulfide cross-linking, but still significantly lower than that of 25 kDa PEI. This could be explained on the basis of GPC analysis results, which showed that there is a big portion of low molecular fraction in the crosslinked products even at high thiolation degree. The particle sizes of the polyplexes were determined by DLS in 150 mM NaCl solution and shown in Table 2. 25 kDa PEI forms the most compact pDNA polyplexes with a size in the range 150-220 nm, and 800 Da PEI forms the loosest polyplexes with a size of about 1000 nm. The cross-linked PEI800-SSY polymers with a moderate thiolation degree (PEI800SS2.6, PEI800-SS3.5, and PEI800-SS4.5) could form compact polyplexes with a size of 200-300 nm at an adequate N/P ratio. In contrast, those with a too low or too high thiolation degree (Y below 2.6 or above 4.5) formed much looser polyplexes with a size above 600 nm. The polyplexes of PEIX-SS3.0-4.0 series (X ) 800, 1800, and 25 000) form small particles with a size below 400 nm at a wide range of N/P ratios. Zeta potentials of the polyplexes were measured in 10 mM NaCl solutions and the results are shown in Table 2. The 25 kDa PEI polyplexes have the highest zeta potential above 35 mV, and the 800 Da PEI polyplexes have the lowest zeta potential of around 10 mV. For PEI800-SSY series, the zeta potentials are negative, showing an incomplete complexation at N/P ratios below 5 (data not shown). With increasing N/P ratio, zeta potentials of the polyplexes rapidly increase and trends to reach a plateau. For PEI800-SS0.4, PEI800-SS1.0, and PEI800SS2.6, it reaches plateaus at N/P ratios between 30 and 40. With the increase of disulfide content in the polymers, the plateau is reached earlier at N/P ratio of about 10. For PEI800-SSY (Y ) 2.6, 3.5, and 4.5), zeta potentials plateau of the polyplexes are all around 25 mV. Surprisingly, PEI1800-SS3.5 and PEI25K-SS4.0 have lower zeta potentials of about 15 mV.

In Vitro Transfection Efficiency. The gene delivery efficiency of the PEIX-SSY series was evaluated by in vitro transfection experiments of pGL3 into HeLa, COS7, 293T, and CHO cells. Branched 25 kDa PEI, which is one of the most widely used highly efficient polycation gene vectors, was used for comparison. The transfection results of PEI800-SSY/pGL3 polyplexes to cultured HeLa, COS7, 293T, and CHO cell lines were presented in Figure 3A. In the PEI800-SSY series, PEI800-SS2.6, PEI800-SS3.5, and PEI800-SS4.5 showed comparable or higher transfection efficiency than 25 kDa PEI at their optimum N/P ratios. Within the range of N/P ratios tested, PEI800-SS3.5 showed maximum efficiencies at N/P ratio of 20 in all four cell lines, achieving nearly 10 times higher luciferase expression as compared with the optimal value of 25 kDa PEI. Comparison of the transfection results in Figure 3 with the size of PEI800-SSY/pGL3 polyplexes listed in Table 2 reveals that the transfection efficiency is in good accordance with the particle size. The polymers with very low thiolation degrees of 0.4 and 1.0 unable to form compact polyplexes had very poor transfection efficiency. The polymers with moderate thiolation degrees of 2.6, 3.5, and 4.5 forming the most compact polyplexes had highest efficiency within the PEI800-SSY series. When the thiolation degree was further increased to a very high level of 5.5 and 7.0, the polyplexes became less compact and the transfection efficiency decreased. Figure 3B shows the transfection results of PEI800-SS3.5, PEI1800-SS3.5, and PEI25K-SS4.0, which have similar thiolation degrees but different molecular weights of raw PEIs, in HeLa, COS7, 293T, and CHO cells. Among them, PEI800-SS3.5 had the highest efficiencies in all four cell lines. PEI1800-SS3.5 had comparable efficiency to 25 kDa PEI in Hela and higher efficiency than 25 kDa PEI in COS7, 293T, and CHO cells. Surprisingly, PEI25K-SS4.0 had much poorer efficiency. Though PEI25K-SS4.0 had similar pDNA retardation capability and cell toxicity compared to 25 kDa PEI, its gene transfection efficiency was much poorer, possibly from the larger sizes and low zeta potentials of its pDNA polyplexes. These results showed that not all raw PEIs with different molecular weights are suitable for cross-linking by disulfide to form degradable polymers possessing an improved capability of gene transfection. Among

Disulfide Cross-Linked Polyethylenimine

the three PEIs of different molecular weights tested (800 Da, 1800 Da, and 25 kDa), the cross-linked polymer prepared from that of the lowest molecular weight (800 Da PEI) gave the best results in all four cell lines tested.

CONCLUSIONS Various amounts of thiol groups were readily introduced to PEIs with different molecular weights using thiirane as a thiolation agent. The thiolated PEIs were transferred to disulfide cross-linked polyethylenimines (PEIX-SSY) by oxidation of the thiol groups with DMSO. Transfection efficiency of the cross-linked polymers is dependent on the thiolation/cross-linking degree Y, and is in good accordance with the particle size of their pDNA polyplexes. Among the PEI800-SSY series with different thiolation degrees prepared from identical raw PEI, the polymers with moderate thiolation degrees of 2.6, 3.5, and 4.5 forming the most compact polyplexes had highest efficiency. The polymers with very low or very high thiolation degrees were unable to form compact polyplexes and had very poor transfection efficiency. A suitable molecular weight of raw PEI is also essential to obtain a highly efficient disulfide cross-linked PEI gene vector. Among the three raw PEIs of different molecular weights tested (800 Da, 1800 Da, and 25 kDa), the cross-linked polymer prepared from the one with the lowest molecular weight (800 Da PEI) gave the best results. Cross-linking of the high molecular weight PEI (25 kDa) led to decreased transfection efficiency.

ACKNOWLEDGMENT This research was financially supported by National Natural Science Foundation of China (20574054), “973 Program” of the Ministry of Science and Technology of China (2005CB623903), and Program for New Century Excellent Talents in Universities (NCET) of the Ministry of Education of China.

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