Biodegradable and Nontoxic Nanogels as Nonviral Gene Delivery

Article pubs.acs.org/bc

Biodegradable and Nontoxic Nanogels as Nonviral Gene Delivery Systems Rajesh Sunasee,† Phanphen Wattanaarsakit,§ Marya Ahmed,† Farahnaz Begum Lollmahomed,† and Ravin Narain*,† †

Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, T6G 2G6, Canada Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Phyathai Road, Pathumwan, Bangkok, Thailand

§

S Supporting Information *

ABSTRACT: The development of polymeric systems with tailored properties as nonviral gene carriers continues to be a challenging and exciting field of research. We report here the synthesis and characterization of biodegradable, temperatureand pH-sensitive carbohydrate-based cationic nanogels as effective gene delivery carriers to Hep G2 cells. The temperature-sensitive property of the nanogels allows their facile complexation of DNA, while the pH-sensitive property allows the degradation of nanogels followed by the release of plasmid in the endosome. The nanogels are synthesized via reversible addition−fragmentation chain transfer polymerization (RAFT) technique and are evaluated for their DNA condensation efficacy. The gene delivery efficacies of these nanogels are subsequently studied and it is found that these cationic glyconanogels can serve as potent gene delivery vectors in hepatocytes. It is found that the gene delivery efficacies of this system are similar to that of branched poly(ethyleneimine), which is used as a positive control. Moreover, these nanogels show desirable properties for systemic applications including low toxicity and degradation in acidic environment.

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INTRODUCTION Transfer of genetic material into cells (both in vitro and in vivo) is crucial for studying gene function as well as conducting gene therapy. The past decade has seen significant progress in this area in order to establish the most desirable method for safe, effective, and target-specific gene delivery.1,2 A variety of synthetic materials including cationic polymers, liposomes, and peptides have been investigated for their gene delivery potential.1−9 Colloidal nanogel carriers have recently gained a lot of interest as novel therapeutic delivery agents in living cells and tissues. These cross-linked polymeric networks provide a core−shell structure, suitable for chemical engineering and for the encapsulation of materials in the core.10−12 The use of nanogels as delivery carriers is desirable, due to their degradable nature, facile encapsulation of materials in the core, release of biomolecules upon an external stimuli, and low toxicity.10−12 However, most of the studies focus on the loading of small molecules in nanogels, followed by their release in living cells, such as proteins, peptides, oligonucleotides, and siRNAs.13−19 It has been shown that chemical cross-linking of cationic particles enables formation of polyplexes when mixed with nucleic acids to increase their stability and gene expression under physiological conditions.19 Hence, cationic nanogels are synthesized and are studied for their gene expression. It has also been reported that encapsulation of large DNA molecules in nanogels leads to significant aggregation; in contrast, siRNA loading is successfully achieved using nanogel systems. © 2012 American Chemical Society

However, these nanogels were not tested for their gene delivery efficacies in vitro.20 The size of nanomaterials plays a critical role in their biological applications.3,4 PEI-based nanogels of varying sizes have been prepared and studied for their gene expression. It is reported that transfection efficacies of these nanogels are dependent on their sizes, and nanogels ranging from 70 to 90 nm show optimum gene expression.21,22 Recently, shell cross-linked kendel-like particles of 10 nm have been prepared to mimic histone-like properties. These nanoparticles containing hydrophobic core and cationic shell are found to efficiently deliver DNA and peptide nucleic acid (PNA) in vitro.23 A variety of carbohydrate-based nanogels are synthesized and studied as gene or drug delivery carriers.11,14,18,24,25 Carbohydrates are important structural and functional units of cell membranes. It has also been shown that incorporation of carbohydrate moieties in gene or drug delivery systems increases their cell viability and serum compatibility and decreases their toxicity.7,8,11 Due to the limited control over the synthesis of polysaccharides-based nanogels, the core cross-linked particles are usually prepared by cross-linking the polysaccharide chains in the presence of DNA.24 To obtain better control of the surface chemistry, size, and hence the gene expression of nanogel-based vectors, cationic Received: June 14, 2012 Revised: August 10, 2012 Published: August 29, 2012 1925

dx.doi.org/10.1021/bc300314u | Bioconjugate Chem. 2012, 23, 1925−1933

Bioconjugate Chemistry

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Scheme 1. Chemical Structures of Monomers (GAEMA, GAPMA, MeODEGM, AEMA and NIPAM), Cross-Linker (CL), Chain Transfer Agent (CTP), and Initiator (ACVA) Used in This Study

temperature, which allowed the collapse of nanogels and entrapment of DNA between cationic cores of nanogels. The pH sensitive property of nanogels further allows the degradation of nanogels at acidic pH (endosomal pH), hence allowing the release of complexed DNA inside the cells. The transfection efficacies of nanogels are studied in Hep G2 cells, using β-galactosidase assay. The cellular uptake of selected nanogel samples are studied using Cy-3′-labeled plasmid. The toxicity post-transfection is obtained using MTT assay.

nanogels with biocompatible moieties can be synthesized via the reversible addition−fragmentation chain transfer polymerization (RAFT) approach.15,26 Earlier reports have shown the synthesis and use of a variety of cationic glycopolymers including diblock polymer, statistical polymers, and hyperbranched polymers for gene delivery applications.7,8,27 Some of these polymers are shown to possess high gene expression along with low toxicity, but they are nondegradable vectors, which is an ideal requirement of a delivery vector for systemic applications. Herein, we report the synthesis and characterization of thermosensitive biodegradable cationic nanogels, as well as the study of their gene expression in Hep G2 cells. The small library of nanogels was characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The rational design of the nanogel systems was guided in part from our recently published work on the synthesis of degradable thermoresponsive poly(2-methacryloyloxyethyl phosphorylcholine), poly(MPC)-based nanogels for protein encapsulation and controlled release.15 In this current system, we opt to have a cationic component in the core and a carbohydrate component on the surface of the nanogel in order to improve the biocompatibility and gene delivery efficacies of the nanogelgene delivery system. The cationic temperature-sensitive core was employed to interact with anionic plasmid DNA at low temperatures (hydrophilic state), followed by the increase in

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EXPERIMENTAL PROCEDURES Materials and Methods. All chemicals were purchased from Sigma Aldrich (Canada) unless otherwise noted. Commercial grade reagents and solvents were used without further purification except as indicated below. Methoxydiethylene glycol methacrylate (MeODEGM, Scheme 1) was passed through a short pad of silica prior to use and Nisopropylacrylamide (NIPAM) was recrystallized three times from hexane. 3-Gluconamidopropyl methacrylamide (GAPMA) 2-gluconamidoethyl methacrylamide (GAEMA), 2-aminoethyl methacrylamide hydrochloride (AEMA), 2,2-dimethacroyloxy1-ethoxypropane, as cross-linker, and 4-cyanopentanoic acid dithiobenzoate (CTP) were synthesized as described according to previous reports.28−32 2-Propanol, HPLC-grade water, acetone, N,N′-dimethylformamide (DMF), dioxane, and hexane were purchased from Caledon Laboratories (Canada). 1926



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elevated temperature (37 °C) then collapses the core and entraps the DNA between the cationic nanogels. Samples were then subjected to electrophoresis on a 1% agarose (135 mV) containing 1.0 μg/mL ethidium bromide in 1× Tris acetate/ EDTA buffer for 25 min. The gels were visualized using UV trans-illuminator (Alpha Innotech; San Leandro, CA) at 300 nm. Characterization of Nanogels. Nuclear Magnetic Resonance. MeODEGM shows thermoresponsive behavior and undergoes coil to globule transition at a lower critical solution temperature (LCST) of 24 °C. This transition was also studied at a molecular level by using variable temperature NMR spectroscopy technique in D2O. Gel Permeation Chromatography (GPC). The number average molecular weight (Mn) and polydispersity (Mw/Mn) of the macro CTAs were determined at room temperature using Viscotek conventional GPC connected to two Waters Ultrahydrogel linear WAT011545 columns (pore size: blend; exclusion limit = 7.0 × 106) and has a Viscotek model 250 dual detector. An acidic buffer of 0.50 M sodium acetate/0.50 M acetic acid was used as eluent. Calibration of GPC was done by six monodispersed poly(ethylene oxide) (PEO) standards (Mp − 1.01 × 103 − 1.01 × 105 g mol−1). Particle Sizes by Dynamic Light Scattering (DLS). Size of nanogels was analyzed using a Viscotek DLS 802 instrument, which is equipped with a He−Ne laser at a wavelength of 632 nm and a Peltier temperature controller. The aqueous nanogel solution was filtered through a 0.45 μm pore size Millipore membrane. Data were obtained at room temperature at an angle of 90° within the temperature range 25−50 °C. Omni Size software was used to record the DLS size data. Particle Sizes and Morphology by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). Size and morphology of nanogels at room temperature were analyzed by TEM on a Philips transmission electron microscope operated at 80 kV and fitted with a CCD camera. A droplet of a properly diluted nanogel solution (in water) was placed on the TEM carbon coated copper grid and allowed to air-dry before it was stained with phosphotungstic acid (2 wt % aqueous solution) for 1 min. The sample was then allowed to dry overnight prior to observation. SEM was performed using a scanning electron microscope FEI XL30 with accelerating voltage of 20 kV. Sample has been prepared by placing an aliquot of a dilute suspension of lyophilized nanogel on a copper grid coated with an amorphous carbon film and let the solvent evaporated. Then, sample was attached to a stub and sputter-coated with Au/Pd. Cell Culture. Hep G2 cells were maintained in low glucose DMEM media supplemented with 10% FBS and 1% antibiotic, in a humidified atmosphere and 5% CO2 at 37 °C. Upon 80% confluency, the cells were trypsinized with 0.25% trypsin with EDTA, were cultured twice a week, and were seeded in 24 well tissue culture plates. Transfection. Hep G2 cells are seeded at the density of 60 000 cells per well, in 24 well tissue culture plates, and are incubated overnight. The media is removed and 100 μL of serum-free OMEM or serum-containing media is added followed by the addition of nanogel-DNA complexes prepared at varying mass ratios in OMEM media. The DNA dose (1.2 μg/well) is kept constant for all the experiments. The cells are incubated for 4 h and complexes containing media are replaced with fresh serum containing media. The cells are allowed to grow for 48 h before their lysis (CHAPS in Sodium Phosphate

Deionized water was obtained from in-house purification system. O-Nitrophenyl β-D-galactopyranoside (ONPG) (enzymatic), ethidium bromide, β-mercaptoethanol, MTT assay kit to determine cell viability, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) were purchased from Sigma Aldrich. Branched PEI (Mw = 25 kDa) was purchased from Polysciences Inc. Cell Culture media Dulbecco’s Modified Eagle Medium (DMEM; high glucose with L-glutamine and sodium pyruvate), penicillin (10 000 U/ mL), streptomycin (10 mg/mL), 0.25% trypsin-EDTA, Dulbecco’s modified Phosphate Buffer Saline (DPBS), and Fetal Bovine Serum (FBS) were from Invitrogen. Micro BCA assay kit was obtained from Fisher Scientific. Gwiz βgalactosidase plasmid was purchased from Sigma Aldrich. Cy3′-DNA labeling kit was from Mirus Bio. Synthesis of poly(GAPMA) and poly(GAEMA) macroCTA by RAFT Polymerization. GAPMA macroCTA was synthesized as previously reported.31 A typical protocol is as follows: The polymerization was achieved at 70 °C, employing ACVA as the radical initiator and CTP as the RAFT CTA. In a 10mL Schlenk tube, GAPMA (1 g, 3.12 mmol) was dissolved in double-distilled deionized water (5 mL) before the addition of 1 mL of DMF solution containing CTP (9 mg, 0. 032 mmol) and ACVA (4 mg, 0.0097 mmol). The solution was degassed by purging nitrogen for 30 min and then placed with stirring in a preheated oil bath at a temperature of 70 °C. After 6 h, the reaction flask was placed in liquid nitrogen to stop the polymerization. The poly(GAPMA) homopolymer was precipitated out in acetone and the residual GAPMA monomer was removed by washing with an excess of methanol. The poly(GAPMA) macroCTA molecular weight (Mn) and molecular weight distribution (Mw/Mn) were found to be 9373 g/mol and 1.17, respectively, by gel permeation chromatography (GPC). Synthesis of Cationic Nanogels by RAFT Polymerization. A typical procedure for the synthesis of poly(GAPMA29-b(MeODEGM-st-CL-st-AEMA)200) nanogel is as follows: The synthesis of the nanogel was achieved at 70 °C, employing ACVA as the radical initiator and poly(GAPMA)29 as the macro-chain transfer agent. In a reaction test tube, poly(GAPMA)29 macroCTA (0.078 g, 0.008 mmol) and AEMA (0.055 g, 0.33 mmol) were dissolved in double-distilled deionized water (3 mL), then MeODEGM (0.25 g, 1.32 mmol), 2,2-dimethacroyloxy-1-ethoxypropane10 as cross-linker (CL) (0.099 g, 0.33 mmol) (20 mol % with respect to total moles of MeODEGM and AEMA) and ACVA (1.2 mg, 0.004 mmol) were dissolved in 2 mL of 1,4-dioxane and added in the above solution. The solution was degassed by purging with nitrogen for 30 min and the reaction was carried out at 70 °C for 24 h. The reaction was then quenched in liquid nitrogen and by exposure to air. The product was purified by dialysis against distilled water for three days using dialysis membrane with a molecular weight cutoff of 12 000−14 000. The nanogel was obtained as a white powder after freeze−drying overnight and was stored in the refrigerator. Nanogels and Plasmid DNA Binding Interaction. The binding ability of β-galactosidase plasmid to nanogels was evaluated by agarose gel electrophoresis. Gwiz-β-galactosidase plasmid (340 ng) was combined with increasing amounts of cationic nanogels (in water) and was incubated at 5 °C for 30 min followed by incubation at 37 °C for 30 min. The low incubation temperature (5 °C) hydrate the cationic core, hence allowing the complexation of DNA with cationic core. The 1927



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GAPMA/GAEMA is chosen based on our previous results, which showed that both GAPMA and galactose bearing polymer (2-lactobionamidoethyl methacrylamide, LAEMA) are well taken up by hepatocytes.8 GAPMA will be an ideal choice for the nanogels, as bulkiness of LAEMA moiety may hinder the interactions of DNA with cationic core, hence causing poor gene complexation and gene expression. Nanogels Synthesis. Macro-CTAs of GAEMA and GAPMA were first synthesized with narrow molecular weight distributions and different degrees of polymerization via the RAFT process (Table 1 and Figure S2, Supporting Information

lysis buffer) (pH = 7.5) followed by two freeze−thaw cycles. The activity of β-galactosidase is detected using β-galactosidase assay. Briefly, ONPG solution (150 μL of 4 mg/mL) is added to 50 μL of lysate volume in the presence of 4.5 μL 100× Mg solution (1.3 μL of β-mercaptoethanol in 400 μL of 0.1 M MgCl2) in 96 well plates and plate is incubated at 37 °C. The yellow color developed is detected using a TECAN Genios Pro microplate reader at 420 nm after 4 h. The total protein content in cell lysate is detected using microBCA assay (Pierce). The development of purple color is read at 570 nm. MTT Assay. The confluent Hep G2 cells are seeded in 24 well tissue culture plates at the density of 80 000 cells per well. The cells are allowed to adhere overnight, and fresh media at varying nanogel/DNA mass ratios (prepared in media, at constant DNA dose 1.2 μg/well) are added in duplicates. The cells are allowed to grow for four hours in the presence of nanogel complexes. The untreated cells and media alone are used as positive and negative controls, respectively. 125 μL MTT dye is then added per well, and plate is incubated for 2 h, followed by the addition of 500 μL of lysis buffer. The plate is read at 570 nm and percent cell viability is determined:

Table 1. GPC Data of Macro-CTAs with Different Target Degrees of Polymerization MacroCTA

Mn,GPC (g/mol)

Mw/Mn

poly(GAEMA63) poly(GAEMA43) poly(GAPMA57) poly(GAPMA29)

19397 13276 18227 9373

1.20 1.19 1.20 1.17

Figure S1−S2). The macroCTAs were then used to copolymerize MeODEGM or NIPAM and AEMA in the presence of the acid-degradable cross-linker, 2,2-dimethacroyloxy-1-ethoxypropane, in water and dioxane using ACVA as initiator (Scheme 2, Supporting Information Figure S3). The nanogels (NGs) obtained by this one-pot protocol15,36,37 were purified by dialysis and lyophilized. Different nanogel compositions were prepared at varying concentrations of cross-linker, and the hydrodynamic sizes were obtained by dynamic light scattering (DLS). It was observed that the sizes of these nanogels could be controlled accordingly (72−198 nm) by varying the cross-linker concentrations (10−20 mol %) and MeODEGM chain length (Table 2, Supporting Information Figure S3). The nanoparticles were characterized by both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) as depicted in Figure 1 to determine the average particle size and morphology. All nanoparticles (NG-A3) were found to be well-defined spherical structures with diameters in the range of 190 to 193 nm in the dry state, which correlate well with the hydrodynamic size obtained by DLS result (198 nm). Furthermore, after freeze− drying, the nanogels could be redispersed in PBS and their sizes remained the same (Supporting Information Figure S4). The above tests indicate that the nanogels possess favorable stability for storage in both solution and solid forms. Poly(MeODEGM) shows thermoresponsive behavior and undergoes coil to globule transition at a lower critical solution temperature (LCST) of 24 °C. Temperature-dependent DLS studies indicated that nanoparticles shrunk from 116 nm at 15 °C to 54 nm at 50 °C (NG-A4) and 185 nm at 15 °C to 78 nm at 50 °C (NG-A2) as shown in Figure 2. The phase transition behavior of MeODEGM core was also studied at a molecular level using variable temperature 1H NMR (VTNMR) spectroscopy. As expected, the 1H NMR peak intensity peak at 3.4 ppm, which corresponds to a methylene group of poly(MEODEGM), collapsed with an increase in temperature (Supporting Information Figure S5). It should be noted that copolymerization of AEMA with MeODEGM during nanogel formation increased the LCST of the final product, due to the incorporation of hydrophilic moiety (AEMA). Nanogel−DNA Binding. An important requirement for an effective gene delivery system is the ability of the nanogels to

%cell viability = (treated cells − negative control) /(positive control − negative control) × 100

Confocal Microscopic Imaging of Cellular Uptake of Polyplexes. Hep G2 cells are seeded in 6 well tissue culture plates containing glass coverslips. The cells are allowed to adhere overnight. The media is changed and fresh serum containing media is added. The nanogels and Cy-3′-labeled DNA (1.2 μg) complexes formulated in OMEM at varying mass ratios are then added in different wells of 6 well plate. After two hours, cells are washed twice with DPBS, and are fixed with 3.7 wt % formalin in DPBS. The cells are rehydrated and are fixed on glass slides. The slides are imaged using Fluoview FV10i Olympus confocal microscope, and samples are excited at 512 nm and are detected at 570 nm, for Cy3′-labeled samples using 60× objective and 1.6× zoom.

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RESULTS AND DISCUSSION Nanogels are “smart” cargo-carrying nanomaterials, which have been used to deliver a variety of biomolecules both in vitro and in vivo.1,2 Recently, cationic nanogels have been synthesized and are used for gene delivery.23,33,34 The incorporation of nontoxic residues in gene delivery vectors is a well-established approach to reduce their toxicity.3−9,11,15,24−26 Phosphorylcholine based nanogels have previously been synthesized by RAFT and are studied for their protein encapsulation and pH-dependent protein release.15,26 In this study, well-defined carbohydratebased cationic nanogels are synthesized by RAFT and are studied for their gene delivery potential. The nanogels bearing cationic thermosensitive core are cross-linked with aciddegradable cross-linker, while the shell of nanogels is fabricated with well-defined glycopolymers. The core of the nanogel consists of two main components, namely, poly(methoxydiethylene glycol methacrylate) (poly(MeODEGM), a well-known thermoresponsive polymer with an LCST of 24 °C,35 and poly(2-aminoethyl methacrylamide hydrochloride) (poly(AEMA), a cationic component that can condense plasmid DNA. The shell of the nanogel consists of a carbohydrate-based component (GAEMA or GAPMA). 1928



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Scheme 2. Synthesis of Cationic Nanogels via RAFT

condense plasmid DNA as shown in Scheme 3. Polycationic materials with high molecular weights and charge densities can bind negatively charged DNA through electrostatic interactions.23 The carbohydrate-based cationic nanogels synthesized were studied for their DNA condensation efficacies. Varying concentrations of nanogels were incubated with β-galactosidase plasmid and the DNA complexations were studied using agarose gel electrophoresis. It was found that low concentrations of nanogels (nanogel:DNA weight by weight ratio ranging from 4 to 20) could effectively complex DNA and produced cationic complexes retained in the well of agarose gels (Supporting Information Figure S6). Although the nanogels could complex DNA at very low w/w ratios, slightly higher amounts of nanogels were required to obtain optimum gene delivery efficacies. The nanogel−DNA complexes formulated were studied for their charge and sizes using zeta

potential instrument and DLS (Figure 3). It is shown that NGA1, NG-A2, NG-A3, and NG-A4 make stable complexes ranging from 100 to 600 nm in diameter at varying w/w ratios. However, NG-A5 produced micrometer-sized (4−5 μm) particles at high w/w ratio (100). The nanogel-DNA complexes are analyzed for their net positive charge in deionized water. It is found that all nanogels maintain net cationic character, when complexed with DNA at high w/w ratios. Though NG-A2 formulates 200 nm complexes with plasmid DNA, these complexes possess low zeta potential (close to 0) at w/w ratio of 60. Gene Expression. The gene delivery efficacies of polysaccharide-based nanogels have been studied. For instance, cross-linked chitosan-based polyplexes were shown to enhance their stability in physiological conditions and hence have high gene expression.24 Herein, carbohydrate-based cationic nano1929



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Table 2. Hydrodynamic Diameter of Nanogels with Varying Chain Length Compositions and Cross-Linker Concentrations nanogel

nanogel composition

NG-A1

GAEMA43-b(MeODEGM-stAEMA-st-CL)200 GAEMA63-b(MeODEGM-stAEMA-st-CL)100 GAPMA29-b(MeODEGM-stAEMA-st-CL)200 GAPMA57-b-(NIPAM-stAEMA-st-CL)200 GAPMA57-b(MeODEGM-stAEMA-st-CL)100

NG-A2

NG-A3

NG-A4 NG-A5

crosslinker (mol %)

hydrodynamic diameter (nm)

polydispersity

20

139 ± 1.0

0.06

10

174 ± 1.5

0.24

20

198 ± 2.0

0.12

20

109 ± 1.1

0.25

20

72 ± 0.8

0.14

Scheme 3. Complexation of pH and Temperature-Sensitive Nanogels with DNA

Figure 3. Size and net charge of polyplexes formed after incubation of cationic nanogels with β-galactosidase plasmid in deionized water.

Figure 1. TEM (A) and SEM (B) images of cationic nanogels (NGA3) synthesized via RAFT.

high as 100 times) is required for gene expression than what is obtained for DNA complexation using agarose gel electrophoresis.8 The DNA nanogel complexes were studied for their gene expression at varying mass ratios and constant DNA dose, in order to determine the optimum conditions for gene expression (Figure S7). It is found that NG-A1, NG-A2, and NG-A3 showed high gene delivery efficacies at varying w/w ratios (Figure 4). The gene delivery efficacies of NG-A1 and NG-A2 were high and comparable to those of branched PEI, in the presence and absence of serum proteins. NG-A3 showed slightly lower gene expression than NG-A2 and NG-A1. It should be noted that although NG-A2 produced neutral complexes as discussed above, these complexes showed high

Figure 2. Hydrodynamic diameter of thermoresponsive cationic nanogels as a function of temperature.

gels of controlled architecture are produced via RAFT and are studied for their gene expression in the presence and absence of serum proteins. Although the complexation of nanogels with DNA occurred at low mass ratios as indicated by agarose gel electrophoresis, high mass ratios were required for gene expression. A series of varying mass ratios were titrated with nanogels to obtain the optimum gene expression. However, it should be noted that complexation of nanogels was done in water. Moreover, this trend is consistent with previous reports which show that high N/P ratio of polymers (sometimes as

Figure 4. Transfection efficacies of nanogel−DNA complexes studied at constant DNA dose (of 1.2 μg/well) and of varying mass ratios of nanogels in the presence (*) and absence of serum, as determined using β-galactosidase assay. (the blue bars indicate the average transfection efficacies, while the red bar indicate standard deviations). 1930



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gene expression; in contrast, NG-A3 produced small particles with high zeta potential, but the transfection efficacy was slightly lower. This difference in gene expression of NG-A2 and NG-A3 might be due to stronger association of DNA with these nanogels, hence causing the slow release of plasmid and low gene expression in vitro. NG-A4 and NG-A5 showed poor gene expression in the presence of serum proteins. Although NG-A5 showed some gene expression in serum free media, it is thought that further aggregation and precipitation of these large complexes might be associated with their low gene expression in serum. NG-A4 showed poor gene expression in the presence and absence of serum proteins. NG-A4 showed efficient complexation of DNA as studied by agarose gel electrophoresis and DLS and zeta potential instruments; poor gene expression of these NIPAM-based nanogels is further investigated using toxicity assay and cellular uptake of complexes. Toxicity Post-Transfection. The toxicity post-transfection of these nanogel−plasmid complexes is analyzed using MTT assay (Figure 5). It is shown that all these carbohydrate-based

Figure 6. Confocal images of Cy-3′-labeled plasmid−nanogel complexes, after 2 h of incubation in Hep G2 in the presence of serum containing media.

indicates the rapid release of DNA in nucleus. NG-A4-based complexes showed poor cellular uptake. The presence of large particles associated with the periphery of cells indicates their aggregation on cell surface and complexes are not taken up even after 2 h of incubation, hence contributing toward their low gene expression. NG-A5 showed lower fluorescence than other nanogel-based complexes. The image shows the presence of small particles associated with the cells; however, these particles are mostly associated with the periphery and are not well internalized. Aggregation of NG-A5 in the presence of serum proteins may have affected their cellular uptake as seen by confocal images, and hence low gene expression was noted. Small particles produced remain associated with cell surface, which do not translate into significant gene expression as discussed above.

Figure 5. Toxicity post-transfection of nanogel−DNA complexes studied at varying mass ratios using MTT assay. (The black bars indicate % cell viabilities, while gray bars indicate standard standard deviations).

cationic nanogels are less toxic than PEI, regardless of mass ratio used for gene expression. Branched PEI showed low cell viability (30%) at low mass ratio (5). It should be noted that NG-A4, although showing poor gene expression, show high cell viability post-transfection, hence, low gene expression of this nanogel was not associated with the cytotoxicity of nanogel. Cellular Uptake. We further studied the uptake of nanogel−DNA complexes in Hep G2 cells using confocal microscopy. To evaluate if the cellular uptake of nanogels was a critical factor in dictating their gene expression, nanogels with distinct physiochemical characteristics and gene profiles were compared. NG-A2 (which produced neutral complexes but showed high gene expression), NG-A4 (which produced small cationic complexes, but no gene expression), and NG-A5 (which produced micrometer-sized particles and showed poor gene expression in the serum containing media) are studied for the uptake of Cy-3′ DNA. Cy-3′ labeled DNA was complexed with nanogels at varying weight/weight ratios, and the uptake of complexes was studied after two hours of incubation with Hep G2 cells in the presence of serum proteins (Figure 6). It is shown that NG-A2 though produce large particles are welluptaken and can accumulate in the nuclei of cells within 2 h of incubation. Moreover, diffused fluorescence in the nucleus

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CONCLUSION The study provides a detailed account on the synthesis of cationic nanogels via RAFT polymerization and their subsequent use as potent gene delivery carriers. Nanogels with a cationic temperature−pH-sensitive core and a permanently hydrophilic glycopolymer shell are fabricated. The nanogels produced are studied for their temperatureresponsive behavior and are characterized using DLS, TEM, and SEM images. All of these cationic nanogels synthesized show high DNA complexation efficacies at relatively low weight/weight ratios, as revealed by agarose gel electrophoresis. The gene delivery efficacies of these carbohydrate-based cationic nanogels are studied. It is found that PEGMA based nanogels showed gene expression comparable to positive control (PEI), along with significantly low toxicities. Although all the nanogels produced show high cell viability, the low gene expression of NG-A4 and NG-A5 was associated with their low cellular uptake as determined from confocal images. The synthesis of biodegradable dual responsive nanogels, their low toxicities, and high gene expression present ideal properties 1931



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required for gene expression vectors. Further studies will be performed to determine the role of amine content, length of glycopolymer chains, and degree of cross-linking on the gene expression profile. Moreover, PEGylated sugar-based nanogels with varying PEG chain length will be prepared and will be studied for their gene expression in vitro.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis of macro-CTAs, GPC data, synthesis of nanogels, VT-NMR spectra, temperature-dependent size change, and agarose gel electrophoresis. 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], Telephone: 1-780-492-1736, Fax: 1-780-492-2881. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge support from Natural Sciences and Engineering Research Council of Canada (NSERC) for the funding of this work.

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REFERENCES

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Biodegradable and Nontoxic Nanogels as Nonviral Gene Delivery

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