Disulfide-Containing Brushed Polyethylenimine Derivative

Article pubs.acs.org/bc

Disulfide-Containing Brushed Polyethylenimine Derivative Synthesized by Click Chemistry for Nonviral Gene Delivery Guangyan Zhang, Jia Liu, Qizhi Yang, Renxi Zhuo, and Xulin Jiang* Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China S Supporting Information *

ABSTRACT: Polyaspartamide-based disulfide-containing brushed polyethylenimine derivatives P(Asp-Az)X-SS-PEIs were synthesized via click chemistry and evaluated as nonviral gene delivery carrier. First, azide-functional poly(aspartic acid) derivatives with various azide-group densities and monoalkyneterminated PEI with disulfide linkages were synthesized. Then, click reaction between the azide-functional poly(aspartic acid) derivative as main chain and the monoalkyne-terminated PEI as branched chain resulted in high-molecular-weight disulfidecontaining brushed PEI derivative. The structure of obtained polymers was confirmed by 1H NMR and FTIR. It was shown that the disulfide-containing P(Asp-Az)X-SS-PEIs were able to bind plasmid DNA and condense DNA into small positive nanoparticles. The reduction-sensitivity of the P(Asp-Az)X-SS-PEI/DNA polyplexes was confirmed by gel retardation assay and dynamic light scattering (DLS) in the presence of DTT. In vitro experiments revealed that the reducible P(Asp-Az)X-SS-PEI not only had much lower cytotoxicity, but also posed high transfection activity (both in the presence and absence of serum) as compared to the control nondegradable 25 kDa PEI. This study indicates that a reducibly degradable brushed polymer P(AspAz)X-SS-PEI composed of low-molecular-weight (LMW) PEI via a disulfide-containing linkage can be a promising gene delivery carrier.

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mimicking the reductive intracellular environment.16,17 Therefore, the introduction of disulfide bridges in the main chain,18−21 in side chains,22 or in the cross-links23−25 of polymers has been widely investigated for the design of polymeric gene delivery carriers. Brushed polymers have recently received much attention due to their unique chemical and physical properties, as well as their potential applications in gene delivery.26−29 Huang et al.30 reported polyaspartamide-based oligo-ethylenimine brushes from poly(L-succinimide) (PSI) via the ring-opening reaction with linear PEI (Mn = 423) as gene delivery vector. Cho et al.31 synthesized brushed PEI for gene delivery by the ring-opening reaction of PSI with branched LMW PEI (with molecular weight of 0.6 kDa or 1.2 kDa). However, there are many active amino groups in one PEI molecule; the ring-opening reactions between PSI and PEI had poor selectivity and should be carefully controlled to avoid cross-linking.30 Because of the high selectivity and high fidelity, “click” chemistry has rapidly become a very popular method for polymer synthesis and modification.32,33 However, no report has been published on reducible brushed PEI via click

INTRODUCTION Gene therapy was viewed as an approach for treating hereditary diseases and acquired diseases such as cancer.1 Cationic nonviral vectors, which interact with negatively charged DNA through electrostatic interactions leading to polyplexes, can enhance cellular uptake efficiency and improve transfection efficiency compared with naked DNA.2 Polyethylenimine (PEI) has a privileged place in nonviral polymeric gene delivery systems due to the superior transfection efficiency of PEI-based polyplexes, probably caused by its proton sponge effect.3,4 However, the high amount of positive charges and their nonbiodegradability result in fairly high cytotoxicity of PEIs, especially for high-molecular-weight (HMW) PEIs, such as 25 kDa PEI.5 Low-molecular-weight (LMW) PEIs such as 1.8 kDa PEI are reported to be less cytotoxic but less effective in gene transfection.6 In order to enhance the transfection efficiency and reduce the cytotoxicity, hydrolytically degradable7−9 and reductively degradable10−13 cationic polymers have been designed for gene delivery. Göpferich et al.14 have published a good review on the reducible disulfide-containing polymers for gene delivery. Reductively degradable polymers with disulfide linkage are usually stable in blood circulation15 and can degrade rapidly (minutes to hours) and release DNA in the presence of reductive 1,4-dithio-DL-threitol (DTT) or glutathione (GSH) © XXXX American Chemical Society

Received: March 16, 2012 Revised: May 8, 2012

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dx.doi.org/10.1021/bc300133r | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry

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Scheme 1. Synthesis Route towards P(Asp-Az)X-SS-PEI

branched PEI with molecular weight of 25 kDa was from Sigma-Aldrich. 2-Bromoethylamine hydrobromide (Shanghai Nanxiang Reagent Co., Ltd., China), 1,1′-carbonyldiimidazole (CDI) (Shanghai Medpep Co., Ltd., China), propargyl alcohol (Wuhan FengFan Chemical Co., Ltd., China), cystamine dihydrochloride (Jiangsu Jintan Medicine Chemical Factory, Jiangsu, China), and 1,4-dithio-DL-threitol (DTT) (SigmaAldrich) were used as received. Plasmid pcDNA3-Luc in TE buffer with concentration of 5.0 mg/mL was from PlasmidFactory (Germany). The purified plasmids were diluted with TE buffer solution and stored at −20 °C. All other chemicals were analytical grade and used as received. Synthesis of Monoalkyne-Functionalized PEI Derivative. A general procedure for the synthesis of monoalkynefunctionalized PEI derivative [PEI1800-(PPA-Cyst)1] has been described in our previous work.34 Here, a slightly modified

chemistry for gene delivery so far. In our previous work, hydrolytically degradable brushed pHEMA−pDMAEMA,9 reduction-responsive cross-linked,25 and hyperbranched34 polyethylenimine derivatives containing disulfide bridges synthesized via click reaction were investigated as gene delivery carriers. In this study, a new strategy for synthesis of disulfidecontaining polyaspartamide-based brushed polyethylenimine derivatives P(Asp-Az)X-SS-PEIs via click reaction is explored, as depicted in Scheme 1. This paper describes the synthesis of the disulfide-containing brushed polyethylenimine derivative and the reduction sensitivity and gene transfection activities of the corresponding polyplexes.

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EXPERIMENTAL PROCEDURES Materials. Branched polyethylenimine (PEI) with molecular weight of 1.8 kDa was purchased from Alfa Aesar, and B



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version has been used: (propargylcarbamate)ethyldisulfide ethyl-1-carbamide-imidazole (PPA-cyst-CI, 0.33 g, 1 mmol) dissolved in chloroform (30 mL) was slowly dropped (around 2 h) into a solution of 1.8 kDa PEI (5.40 g, 3 mmol, 30.75 mmol of primary amine groups, assuming that the primary amine content of the 1.8 kDa PEI is 25% of the total amines) in chloroform (80 mL). Next, the reaction mixture was refluxed using an oil bath (60 °C) for 24 h. The chloroform was removed yielding a yellow liquid (5.61 g, containing imidazole) with a yield of 98%. The structure of the obtained PEI1800(PPA-Cyst)1 was confirmed by 1H NMR and FTIR spectra (Figure S1 and Figure S2). Synthesis of Azide-Functional Poly(aspartic acid). Poly(L-succinimide) (PSI) was synthesized according to the literature35 and characterized by 1H NMR in d6-DMSO (Figure 1D). The molecular weight and the molecular weight

Table 1. Synthesis and Characterization of Azide-Functional Poly(aspartic acid)s sample P(AspAz)33 P(AspAz)65 P(AspAz)100

molar feed ratio of 2azidoethylamine to the polymer unit of PSI

average number of azide groups per 100 polymer unitsa

Mwb (kDa)

PDIb

0.4

33

26

1.40

0.8

65

26

1.41

2

100

27

1.47

a

Calculated from 1H NMR spectra. bEstimated by SEC-MALLS in DMF.

MWDs of P(Asp-Az)X were evaluated by SEC-MALLS using DMF as eluent. The results were summarized in Table 1. FTIR analysis of the resulting product showed an absorption peak at 2100 cm−1 corresponding to the azide group (Figure 2A).

Figure 1. 1H NMR spectra of (A) P(Asp-Az)33 and (B) P(Asp-Az)65 in D2O, (C) P(Asp-Az)100, and (D) Poly(L-succinimide) in d6-DMSO.

distribution (MWD) of the PSI were evaluated by sizeexclusion chromatography-multiangle light scattering (SECMALLS) using DMF as eluent. The weight-average molecular weight and MWD of PSI are 18.0 kDa and 1.29, respectively. 2-Azidoethylamine was synthesized according to the literature.36 The details describing synthesis and characterization of 2-azidoethylamine are provided in the Supporting Information (Figure S3 and Figure S4). PSI (0.28 g, 2.89 mmol) and varying amounts of 2-azidoethylamine were dissolved in 3 mL DMF. The mixture was left to react at room temperature for 4 days. The resulting solution was added to ether slowly and the formed white precipitate was collected and dried in vacuum to obtain azide-functional poly(Lsuccinimide) (PSI-Az). The reaction efficiency of 2-azidoethylamine with PSI determined by 1H NMR analysis was about 80% (Table 1). The remaining succinimide rings of PSI-Az were further reacted at room temperature for 4 days in DMF with excess ethanolamine. The azide group content for the obtained azide-functional poly(aspartic acid)s was determined by 1H NMR analysis (Figure 1). The polymers are denoted as P(Asp-Az)X, where X represents the average number of azide groups per 100 polymer units. The molecular weights and

Figure 2. FTIR spectra of (A) P(Asp-Az)X and (B) P(Asp-Az)X-SSPEI.

Synthesis of Disulfide-Containing Brushed PEI Derivative via Click Reaction. Azide-functional P(Asp-Az)X (50 mg, 1 equiv azide groups) and monoalkyne-functionalized PEI1800-(PPA-Cyst)1 (2 equiv alkyne groups) were dissolved in 10 mL solvent (water, DMF, or the mixture of water and DMF; see Table 2). CuBr (20 mg) was added as catalyst under a nitrogen atmosphere. The reaction mixture was stirred at 50 °C C



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Table 2. Synthesis and Characterization of Disulfide-Containing P(Asp-Az)X-SS-PEIs sample P(Asp-Az)33SS-PEI P(Asp-Az)65SS-PEI P(Asp-Az)100SS-PEI

solvent (the mixture of water and DMF) (v%)

percentage of reacted azide groupa

percentage of grafted PEI1800-(PPA-Cyst)1 to the polymer unit of P(Asp-Az)Xa

WPEI(%)a

Mwb (kDa)

PDIb

100% water

33.9%

11.2%

51

140

1.92

50% water

9.5%

6.2%

37

63

2.15

100% DMF

3.6%

3.6%

25

-

-

a

Calculated from 1H NMR spectra (see the calculation method of the percentage of grafted PEI1800-(PPA-Cyst)1 to the polymer unit of P(Asp-Az)X in Supporting Information). bEstimated by aqueous SEC-MALLS.

SB-G) were used in series. Sodium acetate (NaAc) solution was prepared by dissolving a calculated amount of sodium acetate in reverse osmosis water (0.3 M), and the pH was adjusted to 4.4 with acetic acid.37 A mixture of 70% 0.3 M NaAc solution, pH 4.4, and 30% acetonitrile was used as the eluent at a flow rate of 0.3 mL/min. For SEC in DMF, two chromatographic columns (Styragel HR3, HR4) with a precolumn were used in series. DMF containing 10 mM LiBr was used as the mobile phase at a flow rate of 0.3 mL/min at 30 °C. The eluent was filtrated through a 0.22 μm HPLC filter and degassed prior to use by ultrasound. The data were processed with Astra software (Wyatt Technology). Cell Culture. Human embryonic kidney transformed (293T) cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/L streptomycin at 37 °C under a humidified atmosphere of 95% air and 5% CO2. Cytotoxicity Assay. The cytotoxicity of the different polymers was studied using the MTT assay on 293T cells. The cells were seeded into 96-well plates at a density of 3000 cells/well and 100 μL DMEM containing 10% FBS was added. The cells were allowed to grow at 37 °C under a humidified atmosphere of 95% air and 5% CO2 for 48 h. Thereafter, the medium was replaced with 100 μL of fresh medium, and 100 μL solution of P(Asp-Az)X-SS-PEI or 25 kDa PEI were added (final polymer concentrations ranging from 0.01 to 0.20 mg/ mL). The cells were incubated for 24 h and the medium containing polymer solution was replaced with 200 μL fresh medium. Next, the MTT reagent (20 μL in PBS, 5 mg/mL) was added to each well for further 4 h incubation at 37 °C. Then, the medium was removed and 100 μL of DMSO was added to dissolve the formed formazan crystals. The absorbance at 570 nm was recorded using Multiskan GO microplate spectrophotometer (Thermo Scientific, USA). The relative cell viability (mean ± SD, n = 4) was calculated as cell viability = (ODsample − ODblank)/(ODcontrol − ODblank) × 100%, where ODsample is the absorbance of solution with cells treated by polymers, ODcontrol is the absorbance for untreated cells (without polymer), and ODblank is the absorbance without cells. Formation of P(Asp-Az)X-SS-PEI/DNA Polyplexes and Agarose Gel Retardation Assay. P(Asp-Az)X-SS-PEI based complexes were prepared by addition of 5 μL polymer solution with designed concentrations in HBS (20 mM HEPES, 130 mM NaCl, pH 7.4) to 1 μL of pcDNA3-Luc plasmid DNA (100 ng/μL in TE buffer). The resulting polyplexes dispersions were incubated at 37 °C for 30 min and were then analyzed by electrophoresis in a 0.7% (w/v) agarose gel containing GelRed and in Tris-acetate (TAE) running buffer at 80 V for 60 min. DNA bands were visualized with a UV (254 nm) illuminator and photographed with a Vilber Lourmat imaging system.

for 2 days. The resulting solution was purified by dialysis against water for 1 week (dialysis tube, MWCO 3.5 kDa) to remove unreacted PEI1800-(PPA-Cyst)1 and unmodified PEI, and the formed disulfide-containing brushed PEI derivative P(Asp-Az)X-SS-PEI was collected after freeze−drying. The structure of the P(Asp-Az)X-SS-PEI was analyzed by 1H NMR (Figure 3) and FTIR (Figure 2B). The molecular weights were obtained by aqueous SEC-MALLS as described below.

Figure 3. 1H NMR spectra of (A) P(Asp-Az)33-SS-PEI, (B) P(AspAz)65-SS-PEI, and (C) P(Asp-Az)100-SS-PEI in D2O.

Characterizations. 1H nuclear magnetic resonance (1H NMR) spectra were determined with Mercury VX-300 spectrometer (300 MHz, Varian, USA). Fourier transformed infrared (FTIR) spectra were recorded on a Spectrum one spectrometer (Perkin-Elmer). The molecular weights and the molecular weight distributions of the polymers were evaluated by SEC-MALLS system consisting of a Waters 2690D separations module, a Waters 2414 refractive index detector (RI), and a Wyatt DAWN EOS MALLS detector. For aqueous SEC, two chromatographic columns (Shodex OHpak SB-803 and SB-802.5, Showa Denko, Japan) with a precolumn (Shodex D



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Particle Size and Zeta Potential Measurements. Particle size of the polyplexes was examined in HBS by DLS (dynamic light scattering). The desired amounts of cationic polymers were diluted in 800 μL HBS and mixed with 200 μL HBS containing DNA. The amount of polymer used was calculated on the basis of chosen w/w ratios (weight of polymer to DNA). Zeta potentials of polyplexes were examined in HBG (20 mM HEPES, 5% glucose, pH 7.4). The polyplexes of various w/w ratios prepared as described above were incubated at 37 °C for 30 min. Particle size and ζ-potential were measured by Nano-ZS ZEN3600 (Malvern Instruments) at 25 °C. Reduction Sensitivity of Polyplexes. To 1 mL of dispersions of P(Asp-Az)X-SS-PEI/DNA polyplexes prepared at w/w ratios ranging from 0.2 to 1.4, 100 μL of 100 mM DTT in HBS was added The polyplexes were incubated at 37 °C for 30 min and were then analyzed by electrophoresis in a 0.7% (w/v) agarose gel containing GelRed and in Tris-acetate (TAE) running buffer at 80 V for 60 min. The reduction sensitivity of P(Asp-Az) X-SS-PEI/DNA polyplexes in response to DTT in HBS was also monitored by DLS measurement. Briefly, to a glass cell containing 1 mL P(Asp-Az)X-SS-PEI/DNA polyplexes (w/w ratio of 2.5) in HBS incubated at 37 °C for 30 min, 10 μL of 1.0 M DTT was added. After mixing, the particle size of the polyplexes was monitored at constant time intervals by DLS. The polyplex solution in HBS without DTT was also measured as control. Luciferase Transfection Assay. Transfection activity of polyplexes based on pcDNA3-Luc plasmid, complexed with P(Asp-Az)X-SS-PEI or 25 kDa PEI, was evaluated in 293T cells. The cells were seeded at a density of 70 000 cells/well in a 24well plate. Subsequently, 1 mL of DMEM containing of 10% FBS was added and the cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for 24 h. Next, the medium in each well was replaced by 0.9 mL of serum-free DMEM. The polymer/DNA polyplexes (100 μL, w/w ratios ranging from 2.5 to 30) were then added and incubated with cells for 4 h at 37 °C. The medium was replaced by fresh DMEM containing 10% FBS, and the cells were incubated for 48 h. Thereafter, the medium was removed and 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. Transfection experiments in 293T cells in the presence of serum were also carried out. The protocol was the same as described above, except that the cell medium of DMEM with 10% FBS was used instead of the serum-free DMEM medium. All the transfection assays were carried out in triplicate.

monoalkyne-terminated PEI derivative as branched chain was carried out with CuBr as catalyst, resulting in reducible brushed PEI derivatives with high molecular weight. Monoalkyne-functionalized LMW PEI was synthesized by coupling of (propargylcarbamate)ethy disulfide ethyl 1carbamide-imidazole (PPA-cyst-CI) to PEI following our previous work.34 The structure of the obtained monoalkynefunctionalized PEI derivative PEI1800-(PPA-Cyst)1 was confirmed by 1H NMR and FTIR spectra (SI Figure S1 and Figure S2). It can be seen from the 1H NMR spectrum in Figure S1 that reagent PPA-cyst-CI had reacted quantitatively with PEI. Therefore, no further purification was required for the next click reaction after evaporation removal of the solvent, chloroform. The use of excess PEI was to prevent the formation of the multialkyne-functionalized PEI. In order to investigate the effect of azide group density on the next click reaction, various azide-functional poly(aspartic acid)s were synthesized using different molar feed ratio of 2azidoethylamine to the polymer unit of PSI (Table 1). It can be seen from Table 1 that the azide group content for the obtained azide-functional poly(aspartic acid)s increased with increasing molar feed ratio of 2-azidoethylamine to the polymer unit of PSI. All the reaction efficiencies of 2-azidoethylamine with PSI determined by 1H NMR analysis were close to 80%. It is worth noting that the remaining succinimide rings in azide-functional poly(L-succinimide) (PSI-Az) can react with the amino group of monoalkyne-functionalized LMW PEI, which will cause cross-linking for the next click reaction. Therefore, excessive ethanolamine was used to further react with PSI-Az. The obtained azide-functional poly(aspartic acid)s are denoted as P(Asp-Az)X, where X represents the average number of azide groups per 100 polymer units determined by 1H NMR analysis. It can been seen from Figure 1 that no peak around 5.3 ppm is observed for all the obtained azide-functional poly(aspartic acid)s, which indicates that no unreacted succinimide rings existed in all resulting P(Asp-Az)X polymers. Because the chemical shifts of the methene protons for the coupled 2azidoethylamine overlap with HDO signal at 3.3 ppm in d6DMSO, 1H NMR spectra for both P(Asp-Az)33 and P(AspAz)65 in D2O are shown in Figure 1 and used to calculate the average number of azide groups per 100 polymer units, based on the ratio of the integral between 3.4 and 3.7 ppm (peak e) to the integral from 3.1 to 3.4 ppm (peaks c and d) in Figure 1A,B. However, P(Asp-Az)100 is barely soluble in water; the 1H NMR spectrum of P(Asp-Az)100 was determined only in d6-DMSO (Figure 1C). An absorption peak at 2100 cm−1 corresponding to the azide group is observed for all the FTIR spectra of P(Asp-Az)X polymers (Figure 2A). Thus, the azide-functional poly(aspartic acid)s with various azide group contents has been synthesized successfully. The disulfide-containing brushed PEI derivative was synthesized via click reaction between azide-functional P(AspAz)X as main chain and monoalkyne-terminated PEI1800-(PPACyst)1 as branched chain. The click reaction media were DMF, water, or the mixture of water and DMF (Table 2), because P(Asp-Az)100 is soluble in DMF and not soluble in water. The structure of the obtained disulfide-containing brushed PEI derivative, denoted as P(Asp-Az)X-SS-PEI, was confirmed by 1 H NMR and FTIR analysis. The 1H NMR spectra of P(AspAz)X-SS-PEIs are shown in Figure 3. It can be seen clearly from Figure 3A that three new peaks appear at δ = 7.9 (a, H of the triazole ring), 5.0 (c), and 4.4 (b), indicating the formation of triazole groups by click reaction. The percentage of grafted

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RESULTS AND DISCUSSION Synthesis and Characterization of Disulfide-Containing Brushed PEI Derivative. The three-step synthetic route of disulfide-containing brushed polyethylenimine derivative P(Asp-Az)X-SS-PEI via click reaction is illustrated in Scheme 1. First, one alkyne pendant group was introduced into a lowmolecular-weight (LMW) PEI (1.8 kDa) to obtain monoalkyne-terminated disulfide-containing PEI derivative [PEI1800(PPA-Cyst)1]. Second, azide groups were introduced into the side chains of poly(amino acid) by the aminolysis reaction to poly(L-succinimide) (PSI) to obtain azide-functional poly(aspartic acid) [P(Asp-Az)X]. Then, click reaction between azide-functional poly(aspartic acid) as main chain and E



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PEI1800-(PPA-Cyst)1 to the polymer unit of P(Asp-Az)X was calculated based on the 1H NMR spectra (see the calculation method of the percentage of grafted PEI1800-(PPA-Cyst)1 to the polymer unit of P(Asp-Az)X in Supporting Information). The absorption peaks at 2100 and 2120 cm−1 for P(Asp-Az)X-SSPEIs become weak (Figure 2B), which implies that the azide groups reacted with the alkyne groups. The molecular weights and MWDs of the P(Asp-Az)X-SS-PEIs were determined by aqueous SEC-MALLS. The SEC traces of the P(Asp-Az)33-SSPEI, P(Asp-Az)65-SS-PEI, and 1.8 kDa PEI were supplied in SI Figure S5, which indicates that the starting LMW PEI is negligible in the obtained brushed PEI derivatives P(Asp-Az)XSS-PEIs. The results were summarized in Table 2. The weightaverage molecular weights of P(Asp-Az)33-SS-PEI and P(AspAz)65-SS-PEI are 140 kDa and 63 kDa, respectively, much higher than that of starting polymer P(Asp-Az)X, 26 kDa, which also indicates that PEI grafted into poly(aspartic acid)s. With all 1 H NMR, FTIR, and SEC-MALLS analysis results taken together, it can be concluded that the click reactions between P(Asp-Az)X and PEI1800-(PPA-Cyst)1 were successful. Interestingly, the PEI-grafted percentage decreased with increasing the average number of azide groups per 100 polymer units in azide-functional poly(aspartic acid), as shown in Table 2. For example, the percentage of grafted PEI1800-(PPA-Cyst)1 to the polymer unit of P(Asp-Az)100 was very low (about 3.6%). A possible reason is the solvent DMF used in this click reaction, because DMF is not a good solvent for high-molecular-weight PEI polymer, and a gel-like substance in DMF was observed in this click reaction. In order to investigate the solvent effect on the click reaction, a series of P(Asp-Az)33-SS-PEIs were synthesized under the same click reaction conditions (50 °C, 48 h, using CuBr as catalyst), but in pure DMF, pure water, or mixtures of different DMF/water ratios as solvent, respectively (Table 3). The 1H NMR spectra and the percentage of grafted Table 3. Solvent Effect on the Synthesis of DisulfideContaining P(Asp-Az)33-SS-PEI

Figure 4. 1H NMR spectra of P(Asp-Az)33-SS-PEI in D2O synthesized with pure water, mixtures of different DMF/water ratios (v%), or pure DMF as click reaction solvent.

solvent (the mixture of water and DMF) (v%)

percentage of reacted azide groupa

percentage of grafted PEI1800(PPA-Cyst)1 to the polymer unit of P(Asp-Az)Xa

WPEI(%)a

100% DMF 50% water 75% water 87.5% water 100% water

11.5% 17.9% 20.0% 26.1% 33.9%

3.8% 5.9% 6.6% 8.6% 11.2%

28 37 39 45 51

a

P(Asp-Az)100-SS-PEI, samples P(Asp-Az)33-SS-PEI and P(AspAz)65-SS-PEI were used in the later investigation. Characterization of P(Asp-Az)X-SS-PEI-Based Polyplexes. To be effective as gene vectors, cationic polymer must be able to bind and condense plasmid DNA into nanoparticles. As shown in Figure 5A, when the cationic polymer/DNA weight ratio increased, the mobility of the DNA pcDNA3-Luc was reduced. When the w/w ratio was above 0.8 for P(Asp-Az)33-SS-PEI based polyplexes, the DNA migration

Calculated from 1H NMR spectra.

PEI to the polymer unit of P(Asp-Az)33 for such P(Asp-Az)33SS-PEIs were shown in Figure 4 and Table 3, respectively. It can be seen from Figure 4 that the signal intensities from b (δ = 4.4), c (δ = 5.0), and the protons of PEI-grafted branches increase with the increase of water content in click reaction solvent. The percentage of grafted PEI to the polymer unit of P(Asp-Az)33 increases from 3.8% to 11.2% with the increase of water content in click reaction solvent, as seen in Table 3. By comparison in Tables 2 and 3, very low influence of azidegroup density of P(Asp-Az)X on the PEI-grafted number of P(Asp-Az)X-SS-PEI is observed for both pure DMF and DMF/ water (50/50, v/v) as click reaction solvents, whereas the solvent effect on the click reaction between azide-functional poly(aspartic acid) and the monoalkyne-terminated PEI derivative is obvious. Because of low PEI-grafted number in

Figure 5. Agarose gel electrophoresis retardation assay of P(AspAz)33-SS-PEI/DNA polyplexes at different w/w ratios (A) without DTT and (B) with 10 mM DTT. F



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Reduction Sensitivity of the P(Asp-Az)X-SS-PEI/DNA Polyplexes. The DTT-triggered reduction sensitivity of P(Asp-Az)X-SS-PEI based polyplexes was investigated by agarose gel retardation assay (Figure 5B) and DLS (Figure 7). Free DNA in P(Asp-Az)33-SS-PEI/DNA polyplexes

was almost retarded. According to our previous work, 1.8 kDa PEI was unable to bind DNA even at an N/P ratio of 30 (corresponding to w/w ratio of 3.9).34 This indicates that the obtained brushed PEI derivatives show better DNA binding capability than the starting polymer 1.8 kDa PEI. The particle sizes of P(Asp-Az)X-SS-PEIs/DNA polyplexes were also investigated by DLS as shown in Figure 6A. The

Figure 7. Particle size by DLS of the P(Asp-Az)33-SS-PEI/DNA polyplexes at w/w ratio of 2.5 in the presence and absence of 10 mM DTT in HBS.

incubated with 10 mM DTT (30 min) was visible in Figure 5B (see the difference of Figure 5A and B when the w/w ratios are above 0.8), which indicates that P(Asp-Az)33-SS-PEI based polyplexes released DNA in a reductive environment. The similar results were obtained with 1 h incubation in the presence of 10 mM DTT (SI Figure S6). It can be seen from Figure 7 that the size of P(Asp-Az)33-SS-PEI based polyplexes was stable in HBS without addition of DTT, while it increased rapidly from 250 to 1000 nm over 1 h in the presence of 10 mM DTT, which also confirms the reduction sensitivity of the P(Asp-Az)X-SS-PEI/DNA polyplexes. In Vitro Cytotoxicity. Cytotoxicity of disulfide-containing P(Asp-Az)X-SS-PEIs was determined using MTT assay with 293T cells. Branched PEI (25 kDa) was used as control. As shown in Figure 8, the cytotoxicity of P(Asp-Az)65-SS-PEI was lower than that of P(Asp-Az)33-SS-PEI, because the molecular weight of P(Asp-Az)65-SS-PEI was lower than that of P(Asp-

Figure 6. (A) Particle size by DLS in HBS and (B) zeta potential in HBG of the P(Asp-Az)X-SS-PEI/DNA polyplexes at various w/w ratios. Error bars are standard deviations of 3 measurements.

disulfide-containing P(Asp-Az)X-SS-PEIs prepared from the 1.8 kDa PEI were able to condense DNA into small particles (less than 370 nm). The polyplexes based on P(Asp-Az)33-SS-PEI (with a size of about 150 nm at w/w ratios above 10 in HBS) were much smaller than those based on P(Asp-Az)65-SS-PEI, because the molecular weight of P(Asp-Az)33-SS-PEI was higher than that of P(Asp-Az)33-SS-PEI (Table 2). Zeta potentials of the P(Asp-Az)X-SS-PEI based polyplexes were measured in HBG and shown in Figure 6B. Positive zeta potentials of about 10 mV were observed at w/w ratios above 2.5 for all the P(Asp-Az)X-SS-PEI based polyplexes, whereas polyplexes based on 25 kDa PEI showed higher zeta potential around +32 mV, which indicates that the positive charge density of the P(Asp-Az)X-SS-PEI is lower than that of pure PEI. The reason is that only the grafted PEI part can provide cationic groups and the weight percentages of grafted PEI in P(Asp-Az)X-SS-PEIs are below 51%, as shown in Table 2.

Figure 8. Cytotoxicity of disulfide-containing brushed cationic polymer P(Asp-Az)X-SS-PEI and control 25 kDa PEI to 293T cells after 24 h incubation (mean ± SD, n = 4). G



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of serum. More importantly, the transfection efficiency of P(Asp-Az)33-SS-PEI/DNA polyplexes at w/w ratio of 10 was even higher than that based on branched 25 kDa PEI in the presence of serum. This may be ascribed to its degradability through cleavage of disulfide bonds inside the cells. Thus, the obtained disulfide-containing brushed P(Asp-Az)33-SS-PEI might be suitable for further in vivo gene transfection study.

Az)33-SS-PEI (Table 2). It can also be seen from Figure 8 that both of disulfide-containing P(Asp-Az)X-SS-PEIs had a lower cytotoxicity than 25 kDa PEI, although both the molecular weights were higher than that of 25 kDa PEI. One reason may be that disulfide-containing brushed P(Asp-Az)X-SS-PEIs can be intracellularly degraded, triggered by glutathione after cellular binding and internalization, into relatively low toxicity products, which is in agreement with our previous reports.25,34 Another reason for the low cytotoxicity of disulfide-containing P(Asp-Az)X-SS-PEIs may be their low charge density.38 In Vitro Transfection. The transfection activity of P(AspAz)X-SS-PEI based polyplexes was evaluated in 293T cell using the plasmid pcDNA3-Luc (expressing luciferase) as reporter genes. Branched 25 kDa PEI was used as control. It can be seen from Figure 9 that both P(Asp-Az)X-SS-PEI/DNA polyplexes

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CONCLUSIONS Designed amounts of azide groups were introduced to poly(Lsuccinimide) to obtain azide-functionalized P(Asp-Az)X. Then, the click reaction between the azide-functionalized P(Asp-Az)X as main chain and the monoalkyne-terminated PEI [PEI1800(PPA-cyst)1] was carried out to synthesize disulfide-containing brushed PEI derivatives P(Asp-Az)X-SS-PEIs. The obtained polymer was characterized by 1H NMR, FTIR, and SECMALLS. Very low influence of azide-group density of P(AspAz)X on the PEI-grafted number of P(Asp-Az)X-SS-PEI was observed for both pure DMF and DMF/water (50/50 v/v) as click reaction solvents, whereas the solvent (water content) effect on the click reaction between azide-functional poly(aspartic acid) and monoalkyne-terminated PEI derivative was obvious. It was demonstrated that the disulfide-containing P(Asp-Az)X-SS-PEIs were able to bind plasmid DNA efficiently and condense DNA into small positive nanoparticles. The reduction-sensitivity of the P(Asp-Az)X-SS-PEIs was confirmed by gel retardation assay and DLS in the presence of DTT. In vitro experiments showed that the reducible P(Asp-Az)X-SSPEIs were less cytotoxic, and more effective in gene transfection (both in the presence and in the absence of serum) as compared to the control nondegradable 25 kDa PEI. These results indicate that the obtained disulfide-containing brushed P(Asp-Az)33-SS-PEI might be a promising gene delivery carrier for further in vivo gene transfection.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization of monoalkyne-functionalized PEI derivative [PEI1800-(PPA-Cyst)1] and 2-azidoethylamine; Calculation method of the percentage of grafted PEI1800-(PPACyst)1 to the structure unit of P(Asp-Az)X. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel. +86-27-68755200; fax +86-27-68754509; E-mail address: [email protected]. Notes

The authors declare no competing financial interest.

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Figure 9. Luciferase expression of the P(Asp-Az)X-SS-PEI/DNA polyplexes at different w/w ratios with control 25 kDa PEI in serumfree DMEM (A) and in DMEM medium with 10% FBS (B) in 293T cells.

ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (21174109, 21074100, 20774068), the National Key Basic Research Program of China (2011CB606202, 2009CB930300), and Program for Changjiang Scholars and Innovative Research Team in University (IRT1030).

were able to transfect cells. The transfection efficiency of P(Asp-Az)33-SS-PEI/DNA polyplexes was higher than that of P(Asp-Az)65-SS-PEI/DNA polyplexes in most case. This is due to higher PEI-grafted density of P(Asp-Az)33-SS-PEI than that of P(Asp-Az)65-SS-PEI (Table 2). The transfection efficiency of P(Asp-Az)33-SS-PEI/DNA polyplexes was similar to that based on branched 25 kDa PEI under optimized conditions at N/P ratio of 10 (corresponding to w/w ratio of 1.3) in the absence

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Disulfide-Containing Brushed Polyethylenimine Derivative

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