Multifunctional Hyperbranched Glycoconjugated Polymers Based on

Article pubs.acs.org/bc

Multifunctional Hyperbranched Glycoconjugated Polymers Based on Natural Aminoglycosides Mingsheng Chen,#,†,‡ Mei Hu,#,§ Dali Wang,† Guojian Wang,† Xinyuan Zhu,*,† Deyue Yan,*,† and Jian Sun*,§ †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China ‡ The Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, P. R. China § Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, School of Stomatology, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, P. R. China S Supporting Information *

ABSTRACT: Multifunctional gene vectors with high transfection, low cytotoxicity, and good antitumor and antibacterial activities were prepared from natural aminoglycosides. Through the Michael-addition polymerization of gentamycin and N,N′-methylenebisacrylamide, cationic hyperbranched glycoconjugated polymers were synthesized, and their physical and chemical properties were analyzed by FTIR, 1H NMR, 13C NMR, GPC, ζ-potential, and acid−base titration techniques. The cytotoxicity of these hyperbranched glycoconjugated polycations was low because of the hydrolysis of degradable glycosidic and amide linkages in acid conditions. Owing to the presence of various primary, secondary, and tertiary amines in the polymers, hyperbranched glycoconjugated polymers showed high buffering capacity and strong DNA condensation ability, resulting in the high transfection efficiency. In the meantime, due to the introduction of natural aminoglycosides into the polymeric backbone, the resultant hyperbranched glycoconjugated polymers inhibited the growth of cancer cells and bacteria efficiently. Combining the gene transfection, antitumor, and antibacterial abilities together, the multifunctional hyperbranched glycoconjugated polymers based on natural aminoglycosides may play an important role in protecting cancer patients from bacterial infections.

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as antitumor, antibacterial, and antiviral activity.5−8 Unfortunately, the transfection efficiency of natural cationic polysaccharides is usually disappointing. Even worse, natural cationic polysaccharides are insoluble in physiological conditions, which greatly restricts their applications in disease treatment.9−16 It can be imagined that, if the natural aminoglycosides could be incorporated into the water-soluble polymers, multifunctional gene vectors with high transfection efficiency, antitumor ability, and antibacterial activity might be obtained. By introduction of a branching structure into the polymer backbone, the cationic polymers exhibit compact and globular structures in combination with a great number of various amine groups, which facilitates DNA condensation, gene delivery, and transfection improvement. Very recently, we proposed the construction of highly branched polycations from natural small molecules with plenty of amines such as aminoglycosides.17

INTRODUCTION Combining several useful properties together, multifunctional nanocarriers in gene therapy have drawn much attention.1 Among various multifunctional gene nanocarriers, the combination of biotherapy and chemotherapy is dominant, in which chemical drugs are conjugated or complexed into the gene vectors.2,3 Through the synergism of gene and drugs, the therapeutic efficacy can be significantly enhanced. However, the introduction of antitumor drugs usually weakens the immunity of patients dramatically, which frequently induces serious bacterial infections and thus results in treatment failure. Therefore, the multifunctional gene vectors with antitumor and antibacterial activities are urgently needed in clinical practice. As an abundance of renewable biological resources, natural polysaccharides with aminoglycosides have attracted great attention in recent years.4 Benefiting from the existence of many amino and glycosyl groups, these natural cationic polysaccharides with good biodegradability and low cytotoxicity have been widely used as gene vectors. Moreover, natural cationic polysaccharides exhibit exciting biological activity, such © 2012 American Chemical Society

Received: January 13, 2012 Revised: April 4, 2012 Published: May 17, 2012 1189

dx.doi.org/10.1021/bc300016b | Bioconjugate Chem. 2012, 23, 1189−1199

Bioconjugate Chemistry

Article

Table 1. Reaction Conditions and Results of Michael-Addition Polymerization of MBA and Gentamicina sample

gentamicin (mol)

MBA (mol)

Mn (×103)

Mw (×103)

Mw/Mn

yield (%)

ζ-potential (mV)

D (%)

T (%)

L (%)

DB

HPGM1 HPGM2b HPGM3b

0.01 0.01 0.01

0.015 0.02 0.025

5.4 5.8 6.1

12.4 15.0 15.3

2.7 2.6 2.5

28 35 39

4.7 ± 0.5 9.0 ± 0.5 10.1 ± 0.9

22 21 23

23 25 24

55 54 53

0.45 0.46 0.47

a

D, T, and L represent the fractions of the dendritic, terminal, and linear units, respectively. bThe yield and DB of HPGM2 and HPGM3 were obtained from HPGM2-V and HPGM3-V separately.

For gel permeation chromatography (GPC) measurements, the amino groups of HPGMs were end-capped by benzoyl chloride. Briefly, 0.3 g HPGM (HPGM1, HPGM2, or HPGM3) was dispersed in 30 mL dichloromethane (DCM) with 6 mL triethylamine. Then, 5 mL benzoyl chloride was added slowly, and the solution was stirred at room temperature for 24 h. Subsequently, the liquid was collected. Sufficient hydrochloric acid was added, and the upper aqueous solution was discarded. After addition of a large number of diluted sodium hydroxide aqueous solutions, the upper aqueous solution was discarded. Distilled water was added to wash the remaining solution several times. The DCM solution was added in anhydrous magnesium sulfate, which was filtered and washed with DCM until the color faded. The solution was concentrated by rotary evaporation, and the product was precipitated three times by diethyl ether and dried in vacuum oven overnight. GPC measurements gave the number-average molecular weight (M n ) and molecular weight distribution, which were summarized in Table 1. 1 H NMR (400 MHz, D 2 O, 298 K) δ: 0.80−1.12 (CH3CHCHO-, CCH3); 1.12−2.08 (CHCH2CH); 2.08−2.34 (OCCH2CH2); 2.34−2.48 (CCH); 2.60 (CHCH 2 CH 2 CHO−); 2.80 (CHCH2CH, -OCHCHOHCHO−); 3.08−3.28 (OCCH2CH2); 3.28−3.64 (CHNCH3, CHNHCH3); 4.44 (NHCH2NH); 4.84 (OCHOCHOH). 13C NMR (400 MHz, D2O, 298 K) δ: 28.0−30.0 (NH2CHCH2CHNH-, -NHCHCH2CHNH2, -NHCHCH2CHNH-); 30.0−32.8 (NH2CHCH2CHNH2); 34−34.8 (OCCH2CH2N-); 34.8−37.2 (OCCH2CH2NH); 43.6−45.6 (NHCH2NH); 48.8−50.0 (NH2CHCH2CH2); 51.2−53.2 (−NHCHCH2CH2, -NCHCH 2 CH 2 ); 64.0−66.0 (HOCHCHNHCH 3 ); 66.0−67.2 (HOCHCHNCH3); 71.2−72.8 (-NCH(CH3)CHCH2CH2CHNH-, -NCH(CH3)CHCH2CH2CHN); 74.0−75.4 (−NHCH(CH3)CHCH2CH2CHNH2); 75.4−77.2((-NCH(CH3)CHCH 2 CH 2 CHNH 2 , NHCH(CH 3 )CHCH 2 CH 2 CHNH-, NHCH(CH3)CHCH2CH2CHN) 128.0−129.0 (OCCH2CH2); 129.0−130.0 (OCCH2CH2); 168.0−169, 174−177 (CO). IR (cm−1): 3435 (νas NH2, νas OH), 2946 (νas CH3), 2865 (νs CH2), 1665 (νas carbonyl), 1533 (ν C−N, δNH, δNH2), 1045 (νC−O−C). Polymer Characterization. 1H NMR and 13C NMR spectra were recorded using Bruker Avance III 400 spectrometer in D2O at 298 K. Quantitative 13C NMR spectra were measured to determine the degree of branching with inverse gated 1H decoupling method. Fourier transform infrared (FTIR) spectra were measured by Bruker Equinox55 FTIR spectrometer with KBr pellets in the range 3600−400 cm−1. The ζ-potential values were measured with Zetasizer 2000 (Malvern, U.K.) at 25 °C. HPGMs were dissolved in neutral phosphate buffered saline solution (PBS, pH 7.4) with a concentration of 1.0 mg/mL, and the ζ-potential was assessed three times. The molecular weight and molecular weight distribution of HPGMs were determined by a TSK guadcolumn AW-H (R0064) GPC system (TSKgel SuperAWM-H*2

Based on this new strategy, hyperbranched glycoconjugated polycations with high transfection efficiency, negligible cytotoxicity, and excellent solubility in physiological conditions were prepared, suggesting a possibility for the development of multifunctional gene vectors. In the present work, low toxicity and highly efficient gene vectors were prepared by Michaeladdition polymerization of natural aminoglycoside gentamycin and N,N′-methylenebisacrylamide. Thanks to the incorporation of natural aminoglycoside into the polymer backbone, the resultant hyperbranched glycoconjugated polymers could efficiently inhibit the growth of cancer cells and bacteria. These results indicate that the natural small molecules with biological activity can be linked together to construct versatile nanocarriers for gene therapy. Considering that many useful natural small molecules can be used for the biomedical field, our work provides a new perspective in development of renewable biological resources.

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MATERIALS AND METHODS Materials. Gentamycin sulfate was purchased from Aladdin. N,N′-Methylenebisacrylamide (MBA, 98%) was purchased from J&K China Chemical Ltd.. Branched polyethylenimine (PEI, Mw = 25 kDa, Mn = 10 kDa), 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Dialysis bag (MWCO = 3.5 kDa, soaked and boiled for 10 min separately before use) was purchased from Shanghai Green Bird Development Co. Ltd., China. BCA protein assay kit was purchased from Beyotime (China). Luciferase assay kit was purchased from Promega. All reagents were used as received. Synthesis of Hyperbranched Poly(Gentamicin-MBA)s by Michael-Addition Polymerization. Typically, gentamicin sulfate (5.76 g, 0.01 mol) was dissolved into three roundbottomed flasks with 150 mL saturated NaHCO3 aqueous solution, and then different amounts of MBA (2.36 g, 0.015 mol; 3.1 g, 0.020 mol; 3.93 g, 0.025 mol) were added separately. The reaction solution was stirred at 60, 60, and 45 °C for 3 days, respectively. Crude products were concentrated by evaporation. After dialysis against distilled water for 48 h (MWCO = 3.5 kDa), the residues without or with vinyl groups (HPGM and HPGM-V) were evaporated and dried in a vacuum oven overnight. Yields of HPGM1, HPGM2-V, and HPGM3-V: 1.96 g, 28%; 2.76 g, 35%; and 3.34 g, 39%. Considering that the terminal vinyl groups might induce high cytotoxicity, diethylamine was used to end-cap the excess vinyl groups of HPGM2-V and HPGM3-V. Briefly, HPGM2-V (2.75 g) or HPGM3-V (3.30 g) was dissolved in 100 mL distilled water, and then excessive diethylamine was added. After the reaction solution was stirred at 45 °C for 3 days, the rotary evaporation was carried out to remove the solvent and excess diethylamine. The residues were evaporated and dried in vacuum oven overnight. The final products hyperbranched poly(gentamicin-MBA)s (HPGM2 and HPGM3) were obtained. 1190



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Morphology of HPGM/pDNA complexes was observed by transmission electron microscopy (TEM) with a JEOL 2010 instrument operated at 200 kV. In Vitro Transfection Assay. HPGM was dissolved in PBS (pH 7.4) and filtered through a Millipore filter (pore size: 0.22 μm). HPGM and pDNA were mixed according to the N/P ratios of 0, 5, 10, 20, 30, 40, and 50. Branched PEI was set as controls and N/P ratio was 20 according to the optimized performance. The dosage of pDNA was 0.2 μg in every well. COS-7 cells were seeded at a density of 10 000 cells/well in 100 μL medium (96-well plates), and cells were cultivated for 24 h. The medium was replaced with 50 μL serum-free medium or in the presence of 10% FBS. Then, 50 μL complex solution was added. Cells were incubated for 4 h, and fresh medium with 10% serum replaced the previous. Then, cells were lysed. The transfection activity was evaluated by luciferase assay according to the protocol of Promega. Protein concentration was evaluated according to BCA protein assay kit. Relative light units (RLU) and corresponding protein concentrations were measured by microplate reader system (Biotech). In Vitro Antitumor Evaluation. The 96-well plates were used and HeLa cells were seeded at a density of 9000 cells/well. The cells were cultured for 24 h, and then various concentrations of HPGM solution (HPGM1, HPGM2, and HPGM3) with DMEM were prepared and filtered through a Millipore filter (pore size: 0.22 μm). Appropriate solution was added to get HPGM solution at different concentrations (0.1, 0.2, 0.5, 1, and 2 mg/mL) and cells were incubated for 24 h. The subsequent procedures were the same as those described in the cell viability assay. The cell inhibition efficiency was calculated by the formula as follows:

column, R0091+R0093 Column lot, PMMA calibration) equipped with a refractive index (RI) detector. Dimethylfomamide (DMF) containing 10 mM LiBr was used as the mobile phase at a flow rate of 0.6 mL/min at 40 °C. Evaluation of Buffering Capacity. The buffering capacity of HPGMs was determined by acid−base titration. 18 Specifically, HPGMs were dissolved in 0.15 M NaCl solution to get 1 mg/mL polymer aqueous solution. At the same time, control was set with 0.15 M NaCl solution. The pH value of solutions was adjusted to about 2 with 0.1 M HCl aqueous solution. 50 μL 0.1 M NaOH aqueous solution was dropped into 10 mL assessed solution every time, and the bottle was shaken frequently until a stable reading could be obtained. The pH values were recorded. Cell Culture. Briefly, COS-7 (a kidney cell line of the African green monkey) and HeLa cells (a cell line derived from cervical cancer cells from Henrietta Lacks) were grown at 37 °C with 5% CO2 environment. DMEM (Dulbecco’s modified Eagle’s medium) with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 units/mL) were used. Cells were subcultured every 2−3 days. Cell Viability Assay. The relative cytotoxicity of HPGMs was estimated by standard MTT method. COS-7 cells were seeded in 96-well plates at a density of 9000 cells/well. The cells were cultured for 24 h, and then DMEM was replaced with fresh 200 μL medium. 50 μL of phosphate buffered saline (PBS) containing serial concentrations of HPGM solutions was added in corresponding wells to get final concentrations of 0.1, 0.2, 0.5, 1, and 2 mg/mL. Branched PEI was set as controls. After the cells were incubated for 24 h, stock reagents of 25 μL MTT (5 mg/mL) were added into every well. Four hours later, the medium was removed carefully. 200 μL DMSO was used in every well to dissolve formazan crystals, and the OD490 (optical density at the wavelength of 490 nm) was recorded by BioTech System. The cell survival ratios were converted by the formula as follows:

inhibition efficiency = (1 − [OD490 ]sample/[OD490 ]control) × 100%

Antibacterial Activity Assay. The antibacterial activity was evaluated by the LB broth method.19 The inhibition ratio based on the optical density (OD600) of bacterial suspension was presented as the antibacterial efficiency. Briefly, HPGM aqueous solution was prepared with sterilized LB broth and filtered through a Millipore filter (pore size: 0.22 μm). Then, 5 mL sterilized LB broth was added into a sterile tube with various concentrations of HPGM. The activated Escherichia coli (E. coli) was inoculated into the buret containing the LB solution and incubated with shaking at 37 °C for 12 h. Then, the OD600 values of the bacterial medium were measured by a UV−vis spectrophotometer. The culture with pure LB medium served as the negative control and the bacteria-free solution served as the positive control. The inhibition ratios for HPGM were calculated as follows:

cell viability = ([OD490]sample/[OD490]control) × 100%

In Vitro Degradation of HPGM. In vitro degradation of HPGM was analyzed by NMR technique. Briefly, HPGM was dissolved in different PBS buffer (pH = 7.4, 5.5) to get 10 mg/ mL HPGM aqueous solutions. The solutions were added into tubes and oscillated at 37 °C. At some intervals, 1 mL HPGM aqueous solutions were sucked out, freeze−dried, and analyzed via 1H NMR in D2O. Biophysical Properties of HPGM/pDNA Complexes. A certain amount of pGL3-control vector as a model plasmid DNA (pDNA) was dissolved in PBS (pH = 7.4) solution. To prepare the complex solution of different molar ratios of polymer nitrogen to pDNA phosphorus (N/P ratios), the various concentrations of HPGM aqueous solutions were added separately. The HPGM/pDNA complex solutions of N/ P ratios with 0, 1, 5, 10, 20, 30, 40, and 50 were mixed with appropriate amounts of 6× loading buffer, which were applied in the slots of a 1% agarose gel in sequence. Ethidium bromide (EB) was used to label pDNA. Then, electrophoresis was performed at 100 V for about 1 h. The profile was analyzed with a UV transilluminator (Bio-RAD gel-phase system). HPGM was dissolved in PBS buffer (pH 7.4). A certain amount of pDNA was added to get the aqueous solution of HPGM/pDNA complexes with N/P = 30. After incubation at room temperature for 30 min, a droplet was dropped onto carbon-coated copper grids, dried in the air with natural way.

inhibition efficiency(%) = 100 − 100 × (A t − A 0)/(Ac − A 0)

Here, A0 corresponded to the OD600 values for positive control; At and Ac were OD600 values for HPGM sample and negative control after incubation for 12 h, respectively.

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RESULTS AND DISCUSSION Synthesis and Characterization of HPGMs. As mentioned in previous parts, gentamycin sulfate is a kind of natural aminoglycosides. Unfortunately, cationic small molecule is hard to compress pDNA. To improve its pDNA condensation

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Scheme 1. Preparation and Application of Multifunctional Hyperbranched Glycoconjugated Polymers

Figure 1. 1H NMR spectra of HPGMs (400 MHz, in D2O, 298 K): (a) HPGM1, (b′) HPGM2-V, (b) HPGM2, (c′) HPGM3-V, and (c) HPGM3.

ability, we prepare hyperbranched glycoconjugated polymers by Michael addition between N,N′-methylenebisacrylamide (A2 monomer) and gentamycin sulfate (B8 monomer). The synthetic route to the gentamycin-based hyperbranched polycations and their application is shown in Scheme 1. To promote rapid proton transfer and stabilize charged intermediates, protic solvent (aqueous solution) was chosen to generate branched architecture. Since gentamycin sulfate was used, the base environment was needed to stabilize the resulting negative charges. Therefore, saturated NaHCO3 aqueous solution was adopted as catalyst to increase the reaction activity and then form the highly branched polymers.20

The resulting hyperbranched poly(gentamicin-MBA)s (HPGMs) were characterized by NMR, FTIR, GPC, and ζpotential measurements. Typical 1H NMR spectra of HPGMs are shown in Figure 1. After Michael-addition polymerization, all proton signals become broad in HPGMs. The proton at 4.44 ppm corresponds to −CH2− of the MBA unit (NHCH2NH). Signals of vinyl terminals in HPGM2-V and HPGM3-V are located at 5.68 and 6.12 ppm, which increase with the feeding ratio of the MBA unit. Since the oxidation−reduction potential of vinyl groups induces high toxicity, the terminal double bonds were end-capped by diethylamine. After end-capping, the signals of vinyl groups disappear in HPGM2 and HPGM3 and the methyl signals at 0.95 ppm increase dramatically. Figure 2 1192



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Figure 2. (A) 13C NMR spectra of HPGMs (400 MHz, in D2O, 298 K): (a) HPGM1, (b′) HPGM2-V, (b) HPGM2, (c′) HPGM3-V, and (c) HPGM3; (B) Enlarged and assigned 13C NMR spectra of HPGM3-V.

gives the correspondent 13C NMR spectra. The signals at 128.5/130.0 and 168.5 ppm come from the double-bond and carbonyl groups of acrylamide terminals, which disappear completely after end-capping with diethylamine. As expected, new strong signals at 10.0 ppm assigned to the methyl groups from the diethylamine unit appear. In order to calculate the degree of branching (DB), all structural units of HPGMs including linear, terminal, and dendritic units are listed in Scheme 2. On the basis of quantitative 13C NMR analysis, the DB values of HPGMs could be determined from the following equation:21

vibrations, respectively. The broad stretching vibration around 3435 cm−1 suggests the existence of many hydroxyl and amino groups in HPGMs. The FTIR data suggest that HPGMs are polymerized from gentamycin and MBA monomers. The molecular weights and molecular weight distributions of HPGMs were measured by GPC, and the data are summarized in Table 1. In detail, the number average molecular weights of HPGM1, HPGM2, and HPGM3 are 5400, 5800, and 6100 g/ mol, with the PDI values of 2.7, 2.6, and 2.5, respectively. The real molecular weights of HPGMs should be larger than the values estimated by GPC analysis, because many tertiary amines are present in end-capped polymers and the cationic polymers tend to interact with columns. ζ-Potential analysis demonstrates that HPGMs are positively charged and the charge density is low, giving a chance to further study gene transfer. Buffering Capacity of HPGMs. Through the proton sponge effect, the strong buffering capacity of cationic polymers could enhance the endosomal escape of polyplexes and facilitate their gene transfection.22,23 Therefore, the buffering capacity of HPGMs was assessed by acid−base titration before in vitro transfection. As shown in Figure 4, the pH of NaCl solution changes rapidly with addition of NaOH solution, while all HPGMs show a good pH-buffering capacity. HPGMs are highly branched polymers with some primary, secondary, and tertiary amines, which makes a great contribution to their

DB = (D + T )/(D + T + L)

where L, T, and D represent the fractions of the linear, terminal and dendritic units, respectively. As listed in Table 1, the DBs of these HPGMs are 0.45, 0.46, and 0.47, suggesting the formation of hyperbranched glycoconjugated polymers. FTIR results provide the additional chemical structure information of polymerization products. As displayed in Figure 3, the absorption peaks at 1045 and 1533 cm are assigned to the C−O−C stretching vibration and amino bending vibration, which originate from the gentamycin unit. A strong carbonyl band at 1655 cm−1 confirms the presence of amide bond from MBA unit. The bands at 2865 and 2946 cm−1 are attributed to the symmetric −CH2− and asymmetric −CH3 stretching 1193



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Scheme 2. Schematic Illustration of the Molecular Structure of HPGMs

Figure 3. FTIR spectra of (a) HPGM1, (b) HPGM2, and (c) HPGM3.

Figure 4. Acid−base titration curves of HPGMs, NaCl, and PEI.

buffering capacity. On the other hand, the nitrogen atoms in HPGMs are limited, so the buffering capacity of HPGMs is lower than that of PEI. Considering that the high nitrogen content of polycations frequently results in serious cytotoxicity, the low nitrogen content of HPGMs might reduce the cytotoxicity of materials greatly. Cytotoxicity of HPGMs. Low toxicity is very important for the biomedical application of polymeric materials. Based on the MTT assay, the cell viability of HPGMs was evaluated against COS-7 cells with PEI (Mw = 25 kDa) as a control. Figure 5 demonstrates that HPGM1 has low cytotoxicity, while both HPGM2 and HPGM3 exhibit some cytotoxicity. Compared to the PEI control, all HPGMs show much lower cytotoxicity. As we know, high charge density is one factor that contributes to their cytotoxicity.24,25 Different from the high charge density of PEI, the nitrogen content of HPGMs is low, resulting in the

low cytotoxicity. On the other hand, some terminal vinyl groups appear after the Michael addition of gentamicin and MBA monomers. After end-capping of vinyl groups by diethylamine, the amount of terminal amine increases with the feeding ratio of MBA monomer. Correspondingly, the ζpotential increases from 4.7 ± 0.5 (HPGM1) to 10.1 ± 0.9 (HPGM3), inducing the improved cytotoxicity. Therefore, only HPGM1 with the lowest toxicity was used to evaluate the gene transfection behavior. In Vitro Degradation. As we all know, the degradation of macromolecules decreases the harm and makes them easy to eliminate through the excretion pathway in vivo.26,27 HPGM1 contains many glycosidic and amide linkages, which could be hydrolyzed in acidic, glycosidase, or lactamase environment in vivo.28−30 Figure 6A gives the 1H NMR spectra of the original HPGM1 and the incubated products at pH 7.4 condition for 1194



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ppm at pH 5.5, which reflects the degradation of glycosidic (3.84 ppm) and amide (2.60 and 2.33 ppm) linkages. It can be observed that both glycosidic and amide linkages degrade obviously in 5 days. As a multifunctional carrier, the polymer should accumulate an appropriate concentration to the target position during its long blood circulation, which facilitates the inhibition of tumors and bacteria. In the meantime, the polymer carrier should be biodegradable after the stimulus of specific triggers for security reasons. According to these requirements, the pH-sensitive HPGM1 seems to be a good candidate. Biophysical Properties of HPGM/pDNA Complexes. HPGM1 is a kind of hyperbranched polycation, which could complex with the negatively charged pDNA through the electrostatic interaction. To realize the efficient cell uptake and gene transfection, proper particle size and stability of polycation/pDNA complexes are required. The DNA condensation capability of HPGM1 was studied by agarose gel electrophoresis. Figure 7A shows that pDNA is completely blocked at N/P = 1, suggesting an excellent pDNA condensation capability of HPGM1. To further confirm the gel electrophoresis observation, the TEM measurement was employed to analyze the size and distribution of the HPGM1/ pDNA complexes. It can be observed from Figure 7B that many homogeneous nanoparticles with a diameter smaller than 100

Figure 5. In vitro cytotoxicity of HPGMs with different concentrations to COS-7 cells after 24 h incubation (mean ± SD, n = 3).

different time. It can be found that the 1H NMR spectra are almost unchanged in 51 days, indicating the high stability of HPGM1 at physiological condition. At an acidic environment, the degradation of HPGM1 is accelerated dramatically. Figure 6B gives the variation of chemical shift at 3.84, 2.60, and 2.33

Figure 6. (A) 1H NMR spectra of HPGM1 in D2O at different degradation time cultured at 37 °C under neutral condition (pH 7.4). (B) 1H NMR spectra of HPGM1 in D2O at different degradation time cultured at 37 °C under acidic condition (pH 5.5). 1195



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Figure 7. Biophysical properties of the HPGM1/pDNA complexes. (A) Agarose gel electrophoretic images of the HPGM1/pDNA complexes: N/P ratios of 0, 1, 5, 10, 20, 30, 40, and 50 from left to right in each well; (B) the TEM image of HPGM1/pDNA complexes at N/P = 30.

delivery of HPGM1/pDNA complexes. As we know, glycosyl units are frequently used as signal molecules to interact with the glycoproteins or glycolipids on the cell membranes.35−37 The glycosylation of polycations might facilitate the recognition of HPGM/pDNA complexes to the cells, which makes a contribution of gene transfer and expression. For potential in vivo applications, it is important to evaluate the influence of serum on the transfection capacity. Therefore, the transfection ability of the HPGM1/pDNA complexes in the presence of serum (10% FBS) was also investigated using COS7 cells. Generally speaking, the transfection efficiency in the presence of serum reduces dramatically due to the nonspecific interactions between polycation/pDNA complexes and negatively charged proteins.38−41 However, Figure 8 shows that no significant reduction in transfection efficiency occurs in the presence of serum. Compared to highly positively charged PEI, the low nitrogen content of HPGM1 results in the small zetapotential, which avoids the nonspecific combination of negatively charged proteins. Moreover, the existence of many hydrophilic hydroxyl groups in HPGM1 further improves the stability of polycation/pDNA complexes.42 The high serumtolerant ability of HPGM1 makes it a promising gene delivery vector. In Vitro Antitumor Activity Evaluation. The combination of biotherapy with chemotherapy suppresses tumor growth more effectively than conventional biotherapy alone. Of all the currently employed combination of biotherapy with chemotherapy modalities, the assembly or modification of drugs with macromolecules to prepare the cationic polymer prodrug/ plasmid nanocomplexes is common.2 However, the preparation of such nanocomplexes is usually tedious, which raises the cost of preparation and provides opportunities for chemical pollution. It can be imagined that if the gene vector owns inherent anticancer function, the combination of biotherapy with chemotherapy will become much effective. Considering that many natural cationic polysaccharides exhibit good biological activities, the antitumor ability of HPGM1 was evaluated by MTT assay against HeLa cancer cells. Figure 9 shows that HPGM1 can inhibit the growth of cancer cells. When the final concentration of HPGM1 increases to 0.2 mg/ mL, more than 50% cancer cells die. The inhibitory effect of

nm appear after the complexation of HPGM1 and pDNA. Both gel electrophoresis and TEM results demonstrate that HPGM1 can condense pDNA very well, although the charge density of hyperbranched glycoconjugated polymer is low. In Vitro Transfection Assay. The transfection efficiency was assessed with a luciferase reporter system. Due to its strong proton sponge effect and high charge density, branched PEI containing primary, second, and tertiary amines leads to high transfection efficiency. Therefore, it has been frequently used as a gold standard in polycationic transfection materials.31,32 As shown in Figure 8, the transfection efficiency of HPGM1 in

Figure 8. Luciferase expression of the HPGM1/pDNA at various N/P ratios in COS-7 cells (the expression levels were measured 48 h later, mean ± SD, n = 4).

COS-7 is similar to that of PEI, reflecting the excellent transfection performance of HPGM1. High transfection efficiency in cell lines involves multiple factors of polycations, such as the charge density, the ability to penetrate the cell membrane, and the buffering capacity.33,34 As mentioned above, the buffering capacity of HPGM1 is lower than that of PEI, so the high transfection activity of HPGM1 not only results from the buffering capacity. One possible explanation is that glycosylation of polycations plays an important role in the 1196



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Figure 10. Antibacterial evaluation of HPGM1 against E. coli after culture for 12 h.

Figure 9. Activity of HPGM1 against HeLa cells after incubation for 24 h.

HPGM1 against the HeLa cell is better than that of chitosan (1 mg/mL) from the larvae of the housefly.43 Both HPGM2 and HPGM3 show similar growth inhibition for cancer cells, and the results are given in Figure S3 in the Supporting Information. Despite cationic polysaccharides showing a lower antitumor ability comparing to efficient antineoplastic agents such as doxorubicin, cisplatin, and paclitaxel, there are rarely reports about their side effect and drug resistance, while highly effective antineoplastic agents usually arouse side effect and drug resistance.44 The synergism of gene transfection and antitumor activity of HPGM1 might play an important contribution for the therapeutic efficacy. Antibacterial Activity Assay. Cancer treatments such as chemotherapy and radiation therapy usually result in the low immunity, which frequently induces the bacterial infections. Therefore, the introduction of antibacterial and antitumor properties into the gene vectors becomes very attractive. Considering that the cationic polysaccharides can usually inhibit the growth of bacteria, the antibacterial activities of HPGM1 were evaluated at different concentrations by using the LB broth method. Here, the culture with pure LB medium served as a negative control and the bacteria-free solution served as a positive control. After adding 5 mg/mL HPGM1 into the E. coli LB medium for 12 h, transparent aqueous solution was obtained and no precipitate was observed. Obviously, the growth of the bacteria had been inhibited effectively by HPGM1. The broth absorbance OD600 gives us a more accurate assessment of antibacterial activity, and the result is shown in Figure 10. With the concentration of HPGM1 at 2 mg/mL, 86.1% of E. coli is suppressed. Further increasing the HPGM1 concentration to 5 mg/mL, E. coli is almost completely suppressed. Although many chemical antiseptics such as penicillin exhibit low inhibitory concentration, they usually induce drug resistance and/or serious side effects.45,46 For cationic polysaccharides, both drug resistance and side effects can be avoided. The minimum bactericidal concentrations of many biologically active substances against to E. coli are close to that of HPGM1. For example, minimum bactericidal concentration of scleraldehyde is about 7 mg/ mL.47,48 Therefore, HPGM1 seems to be in the same good antibacterial category as those conventional biologically active substances/polysaccharides. Combining the antibacterial, antitumor, and gene transfection abilities together, the multifunctional hyperbranched glycoconjugated polymer based on

natural aminoglycosides may play an important role in protecting cancer patients from bacterial infections.

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CONCLUSIONS Natural aminoglycosides with biological activity were used as a building block to construct multifunctional hyperbranched glycoconjugated polymers. After polymerization, the antitumor and antibacterial abilities of natural aminoglycosides were endowed into the resultant glycoconjugated polymers. On the other hand, benefiting from their highly branched architecture and degradable polymeric backbone, these cationic glycoconjugated polymers exhibited low cytotoxicity and high gene transfection efficiency. Therefore, multifunctional glycoconjugated gene vectors with high transfection, low cytotoxicity, and good antitumor and antibacterial activities were successfully prepared. The construction of multifunctional gene vectors from natural functional small molecules provides a new opportunity for gene therapy.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary figures including 1H NMR and 13C NMR spectra of gentamycin sulfate. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (X. Z.); [email protected] (D. Y.); [email protected] (J. S.). Tel.: +86-21-34205699. Fax: +86-21-34205722. Author Contributions #

These authors are joint first authors.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is sponsored by the National Natural Science Foundation of China (20974062) and National Basic Research Program 2009CB930400, the Fok Ying Tung Education Foundation (111048), and China National Funds for Distinguished Young Scientists (21025417). 1197



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Multifunctional Hyperbranched Glycoconjugated Polymers Based on

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