Intracellular Delivery of DNA and Enzyme in Active Form Using

Article pubs.acs.org/molecularpharmaceutics

Intracellular Delivery of DNA and Enzyme in Active Form Using Degradable Carbohydrate-Based Nanogels Marya Ahmed and Ravin Narain* Department of Chemical and Materials Engineering and Alberta Glycomics Centre, University of Alberta, 116 St and 85 Ave, Edmonton, AB, T6G 2G6, Canada S Supporting Information *

ABSTRACT: The facile encapsulation of biomolecules along with efficient formulation and storage makes nanogels ideal candidates for drug and gene delivery. So far, nanogels have not been used for the codelivery of plasmid DNA and proteins due to several limitations, including low encapsulation efficacy of biomolecule of similar charges and the size of cargo materials. In this study, temperature and pH sensitive carbohydrate-based nanogels are synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization technique and are studied in detail for their capacity to encapsulate and codeliver plasmid DNA and proteins. The temperature sensitive property of nanogels allows the facile encapsulation of biomaterials, while its acid-degradable profile allows the burst release of biomolecules in endosomes. Hence these materials are expected to serve as efficient vectors to deliver biomolecules of choice either alone or as codelivery system. The nanogels produced are relatively monodisperse and are around 30−40 nm in diameter at 37 °C. DNA condensation efficacy of the nanogels is dependent on the hydrophobic property of the core of the nanogels. The DNA−nanogel complexes are formed by the interaction of carbohydrate residues of nanogels with the DNA, and complexes are further stabilized with linear cationic glycopolymers. The DNA-nanogels complexes are also studied for their protein loading capacity. The degradation of the nanogels and the controlled release of DNA and proteins are then studied in vitro. Furthermore, the addition of a nontoxic, cationic glycopolymer to the nanogel−DNA complexes is found to improve the cellular uptake and hence to improve gene expression. KEYWORDS: carbohydrate-based nanogels, DNA complexation, gene expression, X-gal staining, encapsulation and release of biomolecules

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INTRODUCTION Novel nanomaterials with unique physical, chemical, and biochemical properties are constantly being developed for biomedical applications.1 In the field of gene therapy, nanomaterials are extensively studied and are shown to offer enhanced DNA protection, improved delivery, and high gene expression.1−5 Recently, the development of polymer nanoparticles, specifically nanogels, for gene therapy has gained a lot of attention.6−8 Nanogels are colloidal cross-linked polymeric networks with a distinct core shell morphology for the easy encapsulation of drugs or biomolecules. Stimuli-responsive nanogels are also prepared and are shown to play an important role in delivering a variety of molecules in vitro and in vivo.7,9−12 Moreover, the low toxicity of these nanogels and their biodegradation make them ideal candidates for systemic applications.6−8 Because of their nanometer sizes and facile storage in dry form, nanogels are intensely studied for the encapsulation of small molecules like siRNA, proteins, and chemotherapeutic and imaging agents.9,10,13−15 The delivery of large DNA vectors (plasmids) is usually achieved by the encapsulation of DNA in the core of particles, © 2012 American Chemical Society

during the cross-linking of polymer chains, and DNA is released either by proton sponge effect of cationic particles or by the slow degradation of particles at acidic pH.11,15−17 Recently, Fang et al. has prepared shell cross-linked kendel like particles (SCK) of 10 nm in diameter with histone like characteristics, which are shown to complex peptide nucleic acid as well as DNA and show significant gene expression in Hela cells.14 Some reports show the use of polyethyleneimine (PEI) based nanogels of 40−150 nm in diameter for gene delivery, and it is found that nanogels of 70−90 nm in diameter show optimum (30%) gene expression in mammalian cells.17,18 Recently, cationic glyconanogels of ∼100 nm in diameter are prepared in our group. These nanogels showed gene expression comparable to PEI, along with low toxicities.19 The coencapsulation of gene delivery vectors with therapeutic or imaging agents is an interesting approach but Received: Revised: Accepted: Published: 3160

May 6, 2012 September 7, 2012 September 12, 2012 September 12, 2012 dx.doi.org/10.1021/mp300255p | Mol. Pharmaceutics 2012, 9, 3160−3170

Molecular Pharmaceutics

Article

Scheme 1. Chemical Structure of Monomers Di(ethylene glycol) Methyl Ether Methacrylate (DEGMA), 3-Gluconamidopropyl Methacrylamide (GAPMA), 2-Lactobionamidoethyl Methacrylamide (LAEMA), 2,2-Dimethacroyloxy-1-ethoxypropane (CL, Cross-linker), 4-Cyanopentanoic Acid Dithiobenzoate (CTP), and Initiator 4,4′-Azobis-(cyanovaleric acid) (ACVA) Used for Nanogel Synthesis

ferricyanide(III), fluorescein isothiocyanate (FITC), O-nitrophenyl-β-D-galactopyranoside (ONPG) (enzymatic), 37 wt % formalin, β-mercaptoethanol, an MTT assay kit to determine cell viability, Dulbecco’s phosphate buffer saline (DPBS), di(ethylene glycol) methyl ethyl methacrylate (PEGMA), and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) are purchased from Sigma Aldrich. 2-Propanol, high-performance liquid chromatography (HPLC) grade water, and acetone are purchased from Caledon Laboratories (Canada). Cell culture media, Dulbecco’s modified eagle medium (DMEM) low glucose with L-glutamine and sodium pyruvate, opti-MEM (OMEM), penicillin (10000 U/mL), streptomycin (10 mg/mL), 0.25% trypsin, DPBS, a Quanti IT PicoGreen dsDNA assay kit, a Cy-3′ DNA labeling kit, X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), and fetal bovine serum (FBS) are from Invitrogen. A micro BCA assay kit is obtained from Fisher Scientific. gWiz β-galactosidase plasmid is purchased from Aldevron. N,N′-Methylenebis(acrylamide) is purchased from BioRad Laboratories Inc. βGalactosidase enzyme is from Worthington Biochemical Corporation. BSA is from Promega Corporation. Methods. Synthesis of poly(GAPMA) or poly(LAEMA) macro-CTA by RAFT polymerization. Poly(GAPMA) or poly(LAEMA) macro-CTA are synthesized as previously reported.20,21 In a typical protocol, GAPMA (1 g, 3 mmol), CTP (12 mg, 43 μmol), and ACVA (4 mg, 14 μmol) are dissolved in a water-to-DMF ratio 5:1 in a 25 mL reactor. The solution is degassed by purging nitrogen for 30 min and is placed in an oil bath at 70 °C for 12 h. The polymer obtained is precipitated in acetone and is washed with methanol to remove the residual monomers. The molecular weight and polydispersity are found using aqueous gel permeation chromatography (Viscotek GPC system). The 0.5 M sodium acetate/0.5 M acetic acid buffer is used as eluents at room temperature and at a flow rate of 1.0 mL/min, and pullulan standards (Mw = 500− 404 000 g/mol) are used for calibration. Synthesis of Nanogels by RAFT Polymerization. Poly(LAEMA) macroCTA (0.2 g, 6.7 μmol) and LAEMA (5 mol %, 26 mg) are dissolved in 4 mL of water. PEGMA (0.2 g,

not easily achievable due to various limitations such as the size and charge of the cargo materials.6−8 So far, the codelivery system is limited to the encapsulation of small siRNA or oligonucleotides with therapeutic proteins and imaging agents, and it is found that the encapsulation efficiency is very different for each component to be delivered.15 One possible approach to increase the packaging efficiency of nanogels based gene delivery vectors is to design stimuli responsive nanogels which can exhibit “compartmentalization” that is in different parts of the nanogels. Herein, the synthesis of thermo-responsive, acid degradable nanogels, with hydrophobic core and carbohydrate shell for the codelivery of plasmid DNA and proteins in vitro are reported. The glucose and galactose fabricated nanogels with the temperature sensitive cores are prepared. The structure of monomers, a chain transfer agent, an initiator, and a crosslinker used to make nanogels is shown in Scheme 1. The core is cross-linked at varying densities with an acid degradable cross-linker. The thermosensitive acid degradable nanogels are studied for their DNA complexation efficacy by a PicoGreen assay. The hydrophobic core of nanogels is found to participate in the DNA complexation. The nanogel−DNA complexes are fabricated with cationic glycopolymer, and their gene expression in the presence and absence of serum protein is studied. Furthermore, cellular uptake and degradation of nanogels in hepatocytes is studied as a function of time using a flow cytometer and confocal microscope. The delivery of proteins and enzymes and codelivery of fluorescent proteins with plasmid DNA using nanogel−polymeric system is studied by confocal microscope. Finally, the cell viability of these nanogels is determined using a MTT assay.

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MATERIALS AND METHODS Materials. 2,2-Dimethacroyloxy-1-ethoxypropane (crosslinker), 3-gluconamidopropyl methacrylamide (GAPMA), and 2-lactobionamidoethyl methacrylamide (LAEMA) are synthesized in the laboratory according to previously reported protocols.13,20,21 Protamine, 4,4′-azobis-(cyanovaleric acid) (ACVA), potassium hexacyanoferrate(II) trihydrate, potassium 3161

dx.doi.org/10.1021/mp300255p | Mol. Pharmaceutics 2012, 9, 3160−3170


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protocol. DNA alone in the absence of nanogels and TE (Tris-acetate with EDTA) buffer are used as positive and negative controls, respectively. DLS and Zeta Potential Measurement. gWiz-β-galactosidase plasmid is incubated with nanogels at predetermined w/ w ratios at 4 °C for 3−4 h. The nanogels are collapsed at 37 °C and are either analyzed as such or further incubated with statistical cationic glycopolymer for 15 min. The sizes of complexes are obtained in DPBS and water in the presence and absence of serum proteins. The net charge of complexes is obtained in deionized water in the presence and absence of polymer coating on nanogel−DNA complexes. Gene Expression. The nanogel−DNA complexes at predetermined w/w ratios are formulated as described above, and the complexes are titrated with varying amount of cationic glycopolymers in DPBS buffer. Hep G2 cells are maintained in low glucose DMEM media, supplemented with 10% FBS and 1% antibiotic. Upon 80% confluence, the cells are seeded in 24 well tissue culture plates at density of 90 000 cells/well and are allowed to adhere overnight. The media is replaced with OMEM, followed by the addition of nanogel-complexes in the presence and absence of serum. The cells are incubated for 4 h; the media is then replaced with fresh serum containing media, and cells are allowed to grow for 48 h, before the detection of β-galactosidase activity. The amount of enzyme produced per milligram of protein is detected using O-nitrophenol-β-Dgalactopyranosidase substrate, according to a previously established procedure.22 Delivery of Biomolecules and Degradation of Nanogels. β-Galactosidase enzyme, FITC labeled proteins, and/or Cy-3′-labeled gWiz-β-galactosidase DNA is encapsulated in nanogels as described above. Nanogels are collapsed at 37 °C and centrifuged for 90 min. The supernatant is discarded and protein encapsulated-Cy-3′-labeled-DNA functionalized nanogels are dispersed in warm DPBS. Hep G2 cells are seeded on glass slides mounted 6 well tissue culture plates and are allowed to adhere overnight. The protein and DNA encapsulated nanogels are then added to the cells in the presence of serum containing media and are incubated for 2 h. The media is then replaced, and cells are allowed to grow for another 2, 4, or 6 h. The cells are then fixed on glass coverslips and are mounted on glass slides. The samples are studied using Fluoview FV10i Olympus confocal microscope using 60× objective. Detection of β-Galactosidase Activity in Hepatocytes. Hep G2 cells seeded in 24 well or 6 well tissue culture plates are allowed to adhere overnight. β-Galactosidase enzyme encapsulated nanogels are then added to the cells at varying concentrations and are incubated for four hours. The media is then replaced with fresh serum containing media, and cells are further incubated for 2 h, followed by their lysis or staining with X-gal solution. For X-gal staining, cells are fixed on glass slides and incubated with X-gal stain for 45 min. The glass slides are then imaged using inverted light microscope using 10× objective. For the detection of β-galactosidase activity quantitatively in U/mL, cells are lysed, and protein extracts are analyzed using O-nitrophenol-β-D-galactopyranosidase substrate. The development of yellow color is quantified by reading absorbance at 420 nm using TECAN microplate reader. MTT Assay. The confluent Hep G2 cells are seeded in 96 well tissue culture plates at the density of 80 000 cells per well. The cells are allowed to adhere overnight, and fresh media with varying concentration of nanogels (0.1−5 mg) or polymer (up to 0.13 mM) are added in duplicates. The cells are allowed to

1.1 mmol), cross-linker (CL; 20 mol %, 73 mg, 0.2 mmol), and ACVA (4.0 × 10−4 g, 1.6 × 10−3 mmol) are dissolved in 1 mL of 2-propanol, sonicated, and added to the reaction mixture. The solution is purged with nitrogen for 45 min, and the reaction tube is sealed and placed in oil bath at 70 °C for 24 h. The reaction is stopped by quenching in liquid nitrogen. Nanogels are dialyzed against deionized water and are freezedried to obtain white powder. Synthesis of Statistical Cationic Glycopolymer. Synthesis of cationic glycopolymer P(AEMA17-st-GAPMA20) is obtained according to a previously reported procedure.22 The polymer obtained is analyzed using GPC as described above for its molecular weight and polydispersity. The composition of polymer is determined using Varian 500 1H NMR. Fluorescent Labeling of Proteins. Protamine and BSA) are dissolved in 4% NaHCO3 solution at 1 and 2 mg/mL concentrations, respectively. FITC-DMSO solution (1 mg/mL) is added to protein solution in small portions (final volume 50− 100 μL/mL), during continuous stirring. The solution is incubated in dark overnight. The FITC labeled proteins are dialyzed to remove free FITC. The fluorescent proteins are freeze-dried and stored as orange powder. Fluorescent Labeling of Nanogels. Nanogels are labeled fluorescently by activating hydroxyl groups of carbohydrate moieties, with some modifications to previously reported procedure.23 Briefly, nanogels are dispersed in 4% NaHCO3 solution of pH = 8.5 at 5 mg/mL concentration. FITC-DMSO solution at a concentration of 1 mg/mL is added to aqueous solution of nanogels in small portions during continuous stirring (50−100 μL/mL). The solution is incubated in dark for 4−5 days. The solution is dialyzed against deionized water and freeze-dried to obtain yellow powder. Time-Dependent Cellular Uptake of Fluorescent Nanogels. FITC labeled NG-GAPMA-CL-20 and NGLAEMA-CL-20 are dispersed in serum-free media at the concentration of 3 mg/mL. Hep G2 cells are maintained in low glucose DMEM supplemented with 10% FBS and 1% antibiotics in humidified atmosphere at 37 °C and 5% CO2. Upon 80% confluence cells are seeded in 24 well tissue culture plate at the density of 60 000 cells/well and are allowed to adhere overnight. FITC labeled nanogels are then added at concentration of 1.2 mg/mL per well. The nanogels are incubated with cells for a period of 2, 4, and 24 h. The cells are rinsed with DPBS, trypsinized, and fixed with 3.7 wt % formalin. The cellular uptake of nanogels is studied using Beckman Coulter Quanta SC flow cytometer using the FL1 channel. Encapsulation of Proteins. FITC-labeled bovine serum albumin (BSA), FITC-labeled protamine, and β-galactosidase enzyme are encapsulated in NG-LAEMA-CL-10 and NGLAEMA-CL-20 nanogels. The dried nanogels are incubated with aqueous solution of proteins at concentration of 6 mg/mL of nanogels and varying amounts of proteins at 4 °C for 3−4 h. The nanogels are then collapsed and centrifuged at 37 °C and 17 000 rpm for 90 min. The amount of protein encapsulated in the nanogels is determined using fluorescent plate reader or by β-galactosidase assay. Complexation with DNA. gWiz-β-galactosidase plasmid is incubated with nanogels at varying weight/weight ratios at 4 °C for 3−4 h. The nanogels are then collapsed at 37 °C and centrifuged at 17 000 rpm for 90 min. The supernatant is then collected and detected for the presence of free DNA using Quanti IT PicoGreen assay, according to manufacturer 3162



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Scheme 2. Synthesis of Carbohydrate-Based Nanogels via RAFT

grow for 24 h in the presence of nanogels or polymers. The untreated cells and media alone are used as positive and negative controls, respectively. Then 25 μL of MTT dye is then added per well, and the plate is incubated for 2 h, followed by the addition of 100 μL of lysis buffer. The plate is read at 570 nm, and percent cell viability is determined;

Scheme 3. Schematic for the Synthesis of CarbohydrateBased Nanogels and Their Degradation in the Acidic Environment

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

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RESULTS AND DISCUSSION The synthesis of nanoparticles, specifically nanogels, as delivery systems has received a lot of attention recently. Such nanocarriers show excellent water solubility, long shelf life in dried and solution form, and high biomolecules encapsulation efficacy.1,6−8,24 Moreover, the nanogel surface can be tailored with the molecules of choice to provide biocompatibility and targeting ability.13,25,26 Some carbohydrate-based nanogels are also synthesized and are shown to encapsulate small biomolecules including oligonucleotides.27−29 However, the

use of nanogels for large DNA molecules (plasmid DNA) is restricted due to the limited loading capacity of nanogels. The complexation of DNA using cationic nanogels leads to the formation of large particles due to the aggregation of nanogels.30 The synthesis of cationic nanogels, around 10 nm in diameter, is shown to be better systems for plasmid DNA delivery and hence gene expression.14 Previously, we have 3163



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Table 1. Synthetic Parameters of Carbohydrate-Based Nanogels at Varying Cross-Linker Concentrations and a Comparison of Their Hydrodynamic Diameters at Different Temperatures sample codes

nanogel samplesa

NG-LAEMA-CL-10 NG-GAPMA-CL-10

LAEMA64-b-(PEGMA-st-LAEMA-st-CL)180 GAPMA62-b-(PEGMA-st-GAPMA-stCL)180 LAEMA64-b-(PEGMA-st-LAEMA-st-CL)180 GAPMA62-b-(PEGMA-st-GAPMA-stCL)180

NG-LAEMA-CL-20 NG-GAPMA-CL-20 a

cross-linker (CL) mol %

hydrodynamic size at 15 °C (nm)

hydrodynamic size at 37 °C (nm)

10 10

128 ± 0.4 315 ± 0.38

41 ± 0.2 42 ± 0.2

20 20

95.8 ± 0.25 85.4 ± 0.06

34 ± 0.10 ±0.06

Feed ratio of GAPMA or LAEMA in the nanogels is 5%.

Figure 2. Dynamic light scattering (DLS) and zeta potential measurements of nanogel−DNA complexes in the presence of cationic glycopolymers at varying polymer/plasmid ratios, as shown on the top of bars.

acrylamide (GAPMA) or 2-lactobionamido ethylmethacrylamide (LAEMA) (5 mol %), in the core along with PEGMA is found to improve the stability of the nanogels in buffer solutions. The chemical structures of the monomers, a chain transfer agent, an initiator, and a cross-linker used to make nanogels are shown in Scheme 1. The thermo-responsive nanogels synthesized are then studied for their sizes, and size distributions using dynamic light scattering (DLS) and transmission electron microscopy (TEM; Table 1 and Supporting Information, Figure S1−S2, S3A,C). DLS measurements confirm the thermo-responsive nature of the nanogels. The sizes of nanogels at 37 °C are ∼30−40 nm in diameter. TEM images further show the presence of monodisperse nanogels of ∼40−60 nm in diameter. The size of nanogels is also studied at 15 °C where the core of the nanogels is expected to be hydrophilic and highly hydrated. The sizes of nanogels at low temperature are in the range of 80−300 nm in diameter. The nanogels synthesized at 10 mol % cross-linker concentration show relatively large particle sizes (100−300 nm) than those produced at 20 mol %. The large size of NG-GAPMACL-10 (∼300 nm) at 15 °C is probably due to the aggregation of the nanogels. Chitosan, cellulose, dextran, and hyaluronic acid based stimuli-responsive nanogels has been synthesized in the past and are studied for the encapsulation of biomolecules.15,26,27 However, to the best of our knowledge, this is the first report where thermo-responsive degradable nanogels with a hydrophobic core are synthesized and decorated with a shell of monodisperse synthetic carbohydrate chains in one pot using

Figure 1. Quanti IT PicoGreen assay showing β-galactosidase plasmid DNA condensation efficacy using galactose derived nanogels.

successfully reported the synthesis of cationic glyco-nanogels, which are shown to possess enhanced gene expression.19 Here, we propose a different strategy to produce degradable thermoresponsive nanogels for the delivery of DNA and proteins. Synthesis of Carbohydrate-Based Nanogels. Previously, we have reported the synthesis of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 3-gluconamidopropyl methacrylamide (GAPMA) based degradable and thermo-responsive nanogels by RAFT, and these nanogels are shown to encapsulate proteins of different sizes, and plasmid DNA, respectively, as a function of charge and cross-linking density of the nanogels.13,19 Herein, we report the synthesis of noncationic carbohydrate based nanogels, via RAFT, as shown in Schemes 2 and 3. The nanogels are synthesized with a constant PEGMA (DPn = 180) based core and a glycopolymer shell (DPn ∼ 60), at two different feed cross-linking densities (10 and 20 mol %). The incorporation of glycomonomers, 3-gluconamidopropyl meth3164



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Figure 3. Gene expression of carbohydrate-based nanogels at varying nanogels/plasmid (w/w) ratio and polymer/plasmid (w/w) ratio (top of bars) as shown in parts A in the absence and B in the presence of serum proteins. (Black bars indicate transfection efficiencies, and gray bars indicate standard deviation.)

the RAFT polymerization technique. The nanogels produced are further studied for the encapsulation of proteins and complexation of DNA. Complexation with DNA. The interactions of DNA with carbohydrate residues have been studied by others.31 In this study, carbohydrates functionalized nanogels were studied for their DNA complexation efficacy. DNA was incubated with varying concentrations of nanogels and the DNA condensation efficacy was determined by the PicoGreen assay (Figure 1 and Supporting Information, Figure S4). The DNA complexation efficacy of nanogels is found to be dependent on the type of the nanogels used. For instance, the nanogels with a more hydrophobic core show better DNA complexation efficiency than nanogels with less hydrophobic core. The hydrophobicity of nanogels is dictated by two elements, the length of glycopolymer chains on the surface as well as the cross-linking density of nanogels. The DNA complexation efficiency of nanogels can be summarized by the following trend: NGLAEMA-CL-10 < NG-GAPMA-CL-10 < NG-LAEMA-CL-20 < NG-GAPMA-CL-20. The complexation of nanogels with DNA is further confirmed using TEM images, and the aggregation of nanogels in the presence of DNA is confirmed (Supporting Information, Figure S3 B,D). Although these carbohydrate-based nanogels show complexation with DNA, the preliminary data show relatively low gene expression in Hep G2 cells, possibly due to the aggregation of DNA−nanogel complexes in the media. To

improve the gene expression of these nanogel−plasmid complexes, a statistical cationic glycopolymer P(AEMA17-stGAPMA20) is synthesized via RAFT. The cationic glycopolymer of low molecular weight (Mn = 9.3 kDa; PDI = 1.30) and statistical architecture is chosen to prevent the aggregation of polymer−nanogel−plasmid complexes and to maintain the minimum toxicity of the system.22 The MTT assay shows that P(AEMA17-st-GAPMA20) is highly cell viable (cell viability is 96 ± 1.9% at a 130 μM concentration). The nanogel−plasmid complexes are prepared and further stabilized with P(AEMA17st-GAPMA20) by electrostatic interactions. The sizes and charges of the resultant complexes in the presence and absence of polymer are studied using DLS, TEM, and zeta potential (Figure 2 and Supporting Information, Figure S3). It is shown that polymer−plasmid complexes produce large particles (∼700 nm in diameter) with strong net positive zeta potential values (+35 mV). The nanogel−plasmid complexes are small in size, 50−100 nm in diameter, and the addition of polymer does not increase the size of complexes significantly. This suggests that no aggregation of plasmid−nanogel complexes occurs in the presence of polymer and particles produced are 70−120 nm in diameter. The net charges of plasmid−nanogel complexes range from −9 to 0 mV, as compared to plasmid DNA alone for which net charge is reported to be −40 mV. The addition of the cationic glycopolymer produces nanoparticles with net positive zeta potential values. The net charge of polymer−nanogel−plasmid 3165



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Figure 4. Degradation of FITC-labeled-NG-LAEMA-CL-20 as a function of time in hepatocytes (A) at 2 h, and (B) at 4 h.

PicoGreen assay. Although, GAPMA based nanogels are found to complex DNA at low w/w ratios as compared to LAEMA based nanogels, it is thought that high hydrophobicity and aggregation of GAPMA-based nanogels is a limiting factor in making stable DNA-nanogels complexes, hence contributing toward low gene expression at all studied w/w ratios. In addition, low uptake of GAPMA based nanogels in Hep G2 cells may be a critical factor in their low gene expression, as determined by flow cytometer analysis (Supporting Information, Figure S5). The high uptake of galactose functionalized nanogels in Hep G2 cells is attributed to the specific interaction of galactose residues on the nanogel surface with glycoproteins present on the surface of cells. These proteins, also called asialoglycoprotein receptors (ASGPR), show specific interactions with galactose based nanomaterials.32,33 The gene expression of polymer−plasmid nanogels complexes is further studied in the presence of serum proteins in Hep G2 cells. The stability of gene delivery vectors in physiological conditions is an important factor for their use for in vivo applications. It is found that a slight decrease in gene expression of all DNA−nanogel−polymer based complexes in the presence of serum proteins occurs (Figure 3B). In contrast, polyplexes alone do not show any change in gene expression in the presence of serum proteins. The stability of polymer−DNA−nanogel complexes in the presence of serum proteins is studied by DLS over a period of one hour. DNA− nanogel complexes and polymer−DNA−nanogel complexes do not show any aggregation in serum containing PBS; hence this low gene expression of polymer−DNA−nanogel complexes is

Table 2. Protein Encapsulation Efficiencies of Non-Cationic Galactose-Based Nanogels at Different Cross-Linker Concentrations sample codes NG-LAEMA-CL10 NG-LAEMA-CL10 NG-LAEMA-CL10 NG-LAEMA-CL20

protein

protein encapsulated (in μg/mg of nanogels)

protamine

52

BSA

83

βgalactosidase βgalactosidase

75 (mU/mg) 158 (mU/mg)

complexes is lower than polyplexes alone, possibly due to the passivation of surface charge by the carbohydrate shell of nanogels. Gene Expression. The nanogel−plasmid complexes synthesized are studied for gene expression in Hep G2 cells in the presence and absence of serum proteins as shown in Figure 3. In general, the nanogels, NG-LAEMA-CL-10 and NG-LAEMA-CL-20, show high gene expression, as compared to nanogels NG-GAPMA-CL-10 and NG-GAPMA-CL-20. The gene expression of NG-LAEMA-CL-20 is higher than polyplexes alone and is comparable to linear poly(ethyleneimine) (PEI), which is used as positive control for gene expression.22 NG-LAEMA-CL-20 show high gene expression as compared to NG-LAEMA-CL-10, possibly due to high DNA condensation efficacy of NG-LAEMA-CL-20 as revealed by the 3166



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microscope. The confocal images show the presence of fluorescent particles in cells after 2 h (Figure 4A). However, the removal of media and culturing of cells for another 2 h causes significant reduction in the number of particles in Hep G2 cells as shown by confocal microscope image 4B. This reduction in number of fluorescent particles is thought to be associated with the degradation of nanogels in the cells. It should be noted that fluorescent nanogels around the cell periphery are intact, and significant decrease in number of particles inside the cells is observed. An important property of nanogel-based vectors is the encapsulation of biomolecules like proteins and chemotherapeutic agents and their release in the presence of external stimuli.9,10,13−15,30,35 The protein loading ability of nonionic carbohydrate based nanogels is studied by encapsulating cationic and anionic proteins (FITC-labeled protamine and BSA, respectively) in the core of galactose (LAEMA) based nanogels. The encapsulation efficiency of these proteins is shown in Table 2. FITC-BSA encapsulated nanogels are then studied for their uptake in Hep G2 cells as a function of time as discussed above (Figure 4C,D and Supporting Information, Figure S6). It is found that, at time period of 2 h, FITC-BSA encapsulated nanogels exist as intact particles in the cells; however, a decrease in particles number over a period of time is observed and is most likely associated with the degradation of nanogels, as discussed before. The coencapsulation of proteins (protamine or BSA) and DNA has also been achieved and will be discussed in detail later. Determination of Enzymatic Activity in Vitro. The activity of encapsulating proteins after its delivery in mammalian cells is critical in estimating the success of a therapy vector. To determine, if nanogel-encapsulated biomolecules maintain their activity, β-galactosidase enzyme is encapsulated in the core of galactose (LAEMA) based nanogels, and its encapsulation efficiency in terms of milliunits (mU/mg of nanogels) is determined (Table 2). NG-LAEMA-CL-20 show high encapsulation efficacy of βgalactosidase enzyme, as compared to NG-LAEMA-CL-10. These results are consistent with previous work, where MPCbased nanogels synthesized at different cross-linker concentrations were studied for their protein encapsulation efficiency.13 The enzyme loaded nanogels are then used as delivery system in Hep G2 cells, and the amount of active βgalactosidase enzyme delivered is determined using X-gal staining qualitatively (Figure 5). Figure 5A shows the delivery of β-galactosidase enzyme in cells using galactose-based nanogels. β-Galactosidase enzyme itself is impermeable to cell membrane and do not show any activity by X-gal staining (Figure 5B). These results are in agreement with previous literature, where gold nanoparticles are used as vehicle to deliver β-galactosidase enzyme in Hela cells.24 To quantify the amount of β-galactosidase delivered in the cells, cell lysates obtained from enzyme encapsulated nanogels are treated with O-nitrophenyl-β-D-galactopyranosidase substrate. The development of yellow color (an indication of β-galactosidase activity) is read at 420 nm and is converted into mU/mL (Figure 5C). A 2-fold increase in β-galactosidase activity is observed after treatment with enzyme loaded nanogels, as compared to untreated cells. The cells treated with NG-LAEMA-CL-20 encapsulated β-galactosidase enzyme show a high delivery of enzyme (in mU/mL), as compared to NG-LAEMA-CL-10,

Figure 5. Delivery of β-galactosidase enzyme in hepatocytes using galactose derived using X-gal stain (A) β-galactosidase encapsulated NG-LAEMA-CL-10, (B) β-galactosidase enzyme alone, and (C) determination of β-galactosidase activity in Hep G2 in U/mL after incubation with galactose derived nanogels.

not due to the aggregation of complexes in the presence of serum proteins (data not shown). We attribute this decrease in gene expression of plasmid−nanogel complexes with the low protection ability of plasmid−nanogel complexes in the presence of serum proteins. Due to their high cellular uptake, gene expression and stability in media, galactose-based nanogels NG-LAEMA-CL-10 and NG-LAEMA-CL-20 are further studied for the encapsulation and release of proteins in mammalian cells. In Vitro Degradation of Nanogels and Encapsulation of Proteins. The design of stimuli-responsive systems for the encapsulation and release of biomolecules in vitro is wellstudied.11,13,16,34 However, to the best of our knowledge, there are no reports so far which show the degradation of pH responsive nanoparticles, in mammalian cells. The behavior of particles under physiological conditions may vary as compared to in vitro studies. To study the degradation of nanogels and release of biomolecules in mammalian cells, galactose based nanogels are labeled with FITC and are allowed to interact with Hep G2 cells for 2 h (Figure 4 and Supporting Information, Figure S5). The incubation time of particles with mammalian cells is set to be 2 h, as our previous studies indicate that nanogel degradation occurs after 100 min in buffer of pH 4.513 and flow cytometer analysis show significant uptake of nanogels within 2 h of incubation time. The uptake of FITC-labeled nanogels increases slowly if the cells are incubated for longer period of times in nanogels containing media (Supporting Information, Figure S5). In this study, the treatment is removed after 2 h; the media is replaced with fresh serum containing media, and the cells are allowed to grow for another 4, 6, and 10 h. The cells are fixed at 2, 4, 6, and 10 h intervals, and the presence of fluorescent particles in Hep G2 cells is studied using a confocal 3167



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Figure 6. Confocal images of FITC-labeled-NG-LAEMA-CL-20 and Cy-3′-labeled complexes in the presence of statistical cationic glycopolymers (A) at 2 h and (B) at 4 h. Co-delivery of FITC-BSA encapsulated NG-LAEMA-CL-10 and Cy-3′ labeled DNA complexes in the presence of statistical cationic glycopolymers (C) at 2 h and (D) at 4 h.

which is consistent with high encapsulation of β-galactosidase enzyme in NG-LAEMA-CL-20 sample (Figure 5C). Cellular Uptake of Polymer Plasmid-Nanogel Complexes. The interaction of galactose-based nanogels with plasmid DNA in the presence of statistical cationic glycopolymers in Hep G2 cells is studied using confocal microscopy. FITC-labeled-NG-LAEMA-CL-20 is surface-functionalized with Cy-3′ labeled plasmid followed by the addition of P(AEMA17-st-GAPMA20) at polymer/plasmid ratio of 8. The uptake of complexes and their degradation is studied as a function of time as revealed by confocal microscopic images (Figure 6A,B). The fluorescent images of Cy-3′ labeled plasmid overlaid with FITC-labeled nanogels confirm the complexation of plasmid DNA with nanogels in cells even after two hours of incubation time (as apparent by yellow dots in images) (Figure 6A). Further incubation of cells in fresh media leads to significant reduction in number of fluorescent particles in cells and is attributed toward the degradation of particles inside the cells. The yellow particles associated with periphery of the cells stay intact, even after four hours of incubation, indicating that

pH dependent degradation is the main mechanism to release DNA from the complex, along with endosomal escape aided by cationic glycopolymer. Co-delivery of Protein and DNA. The codelivery of biomolecules (DNA and proteins) is then achieved and is studied as a function of time. FITC-BSA encapsulated NGLAEMA-CL-10 are functionalized with Cy-3′-labeled plasmid, and after addition of P(AEMA17-st-GAPMA20) at polymer/ plasmid ratio of 8, the uptake of the complexes is studied in Hep G2 as shown in Figure 6C,D. Confocal microscopic images show the presence of orange particles inside the cells (in overlay mode), indicating the codelivery of BSA and plasmid in the cells. (The formation of orange particles as compared to yellow particles observed earlier is associated with the fact that in the case of FITClabeled nanogels large carbohydrate chains are labeled and their strong interaction with Cy-3′ labeled DNA produce yellow particles, in contrast encapsulation of FITC-BSA in the core of nanogels and its interaction with plasmid show orange color particles). Similarly, protamine encapsulated nanogels function3168



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Figure 7. Cell viability of carbohydrate based nanogels as a function of cross-linker density as determined by an MTT assay.

expression is found to be relatively low. The addition of a nontoxic, cationic glycopolymer is found to improve the cellular uptake and gene expression of DNA−nanogel complexes. The galactose-based nanogels are recognized as remarkable systems for the codelivery of plasmid and proteins in Hep G2 cells. Under all studied concentrations, it is found that NG-LAEMACL-20 can efficiently encapsulate proteins and condense plasmid DNA and as well show high gene expression in Hep G2 cells, with low cytotoxicity.

alized with Cy-3′-labeled plasmid (in the absence of polymer layer) are also studied, and it is found that protamine encapsulated nanogels show high uptake of plasmid−nanogel complexes in the cells, as compared to nanogels alone (Supporting Information, Figure S7). However, gene expression is only slightly increased using protamine−nanogel complexes (data not shown). Toxicity of Nanogels. The toxicity of nanogels as a function of cross-linked concentration is studied using MTT assay in Hep G2 cells. It is found that regardless of type of carbohydrate used for the nanogel synthesis, the toxicity of nanogels is dependent on their cross-linker feed concentration. In general, NG-LAEMA-CL-10 and NG-GAPMA-CL-10 are more toxic than NG-LAEMA-CL-20 and NG-GAPMA-CL-20 (Figure 7). These results are in agreement with previous literature where the cell viability of poly(ethyleneglycol) (PEG) stabilized nanogels synthesized at different cross-linker concentration is studied.26 Low cell viability of nanogels synthesized at low cross-linker concentrations is associated with the low surface functionalization and exposure of the hydrophobic corona to the cells.25

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ASSOCIATED CONTENT

* Supporting Information S

DLS data, flow cytometer analysis, PicoGreen assays, TEM images of nanogels, and confocal images. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*University of Alberta, Department of Chemical and Materials Engineering and Alberta Glycomics Centre, 116 St and 85 Ave, Edmonton, AB, T6G 2G6, Canada. E-mail: [email protected]. Tel.: 780 492 1736. Fax: 780 492 2881.

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CONCLUSION The study provides a detailed description on the synthesis of carbohydrate based nanogels at two different cross-linker concentrations. It is found that all of the nanogels produced are ∼30−40 nm in diameter at 37 °C. The DNA condensation efficacy of the nanogels is dependent on the hydrophobic core of the nanogels. Although, complexation to DNA is achieved with the temperature sensitive nanogels, and the gene

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by funding from Natural Sciences and Engineering Research Council of Canada (NSERC). The authors would like to thank Dr. Hasan Uludag and his laboratory members for the use of flow cytometer instrument. 3169



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