Uronic acids functionalized polyethyleneimine (PEI)–polyethyleneglycol (PEG)-graft-copolymers as novel synthetic gene carriers

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Biomaterials 27 (2006) 2302–2312 www.elsevier.com/locate/biomaterials

Uronic acids functionalized polyethyleneimine (PEI)–polyethyleneglycol (PEG)-graft-copolymers as novel synthetic gene carriers Sabine I. Weissa,1, Nathalie Sieverlingb,c,1, Maren Niclasenb,d, Christof Mauckscha,e, Andreas F. Thu¨nemannd, Helmuth Mo¨hwaldc, Dietrich Reinhardta, Joseph Roseneckera, Carsten Rudolpha,e, a Ludwig-Maximilians-University, Pediatrics, Lindwurmstr. 2A, 80337 Munich Germany Fraunhofer Institute for Applied Polymer Research, Geiselbergstr. 69, 14476 Golm, Germany c Max Planck Institute of Colloids and Interfaces, Am Mu¨hlberg 1, 14476 Golm/Potsdam, Germany d Federal Institute for Materials Sciences and Testing, Richard-Willsta¨tter-Str. 11, 12489 Berlin e Free University of Berlin, Department of Pharmacy, Takustr. 3, 14166 Berlin, Germany b

Received 5 July 2005; accepted 9 November 2005

Abstract In this study, we investigated galacturonic (GalAc)- and mannuronic (ManAc) acids as novel targeting ligands for receptor-mediated gene delivery. GalAc and ManAc were coupled to either polyethyleneimine (PEI) or PEI–polyethyleneglycol (PEG). Furthermore, lactobionic acid (LacAc), which comprises a GalAc-related carbohydrate ring, was coupled to each of the polymers through its openchain gluconic acid moiety. The molar mass distributions of the polymers were characterized by analytical ultracentrifugation and size exclusion chromatography. PEI-conjugate–pDNA complexes were transfected into HepG2-, HeLa-, and 16HBE14o
-cells. Gene expression mediated by GalAc- and LacAc-functionalized PEI-conjugates was lower than for PEI. In contrast, gene expression mediated by ManAc-functionalized PEI-conjugates was up to three orders of magnitude higher than for the other tested PEI-conjugates, in particular for negatively charged gene vectors at low N/P ratios, independent of the cell line. Pre-incubation of cells with an excess of ManAc before transfection significantly inhibited transfection rates only for ManAc-functionalized PEI-conjugates. Coupling of methyla-D-mannuronic acid to PEI resulted in significantly lower transfection rates than for ManAc-PEI based complexes. Together with fluorescence microscopy images of fluorescein-labelled ManAc-functionalized dextrans and FACS analyses of cells, these results demonstrate that receptor-mediated endocytosis of ManAc–PEI-conjugate–pDNA complexes via ManAc-specific receptors was involved in gene transfer. In conclusion, ManAc-modification of PEI-polymers represents a novel strategy for receptor-mediated gene delivery which could be promising for in vivo application. r 2005 Elsevier Ltd. All rights reserved. Keywords: Gene therapy; Gene transfer; Polyethylene oxide; Nanoparticle

1. Introduction Gene transfer in medical therapy has to ensure efficient transgene expression in target cells, but additionally has to Corresponding author. Current address: Division of Molecular Pulmonology, Department of Pediatrics, Ludwig Maximilians University of Munich, Lindwurmstr. 2a, D-80337 Munich, Germany. Tel.: +49 89 5160 7524; fax: +49 89 5160 4421. E-mail address: [email protected] (C. Rudolph). 1 S.I. Weiss and N. Sieverling have equally contributed to this work.

0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.11.011

be safe and readily applicable. Furthermore, the ideal gene delivery systems should provide effective and targeted transfer of therapeutic genes into the cell type of choice in vivo. The use of nonviral vectors, such as cationic polymers and lipids, is attractive for in vivo gene delivery because it is technically less demanding than using viral systems and lacks some of the risks inherent in the latter [1]. Among them, polyethyleneimine (PEI) is one of the most extensively investigated polycations for gene delivery. It has been shown that PEI mediates effective gene transfer in vitro and in vivo [2–7] by condensing plasmid DNA (pDNA) into small

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particles called polyplexes and leads to high transfection efficiency due to its so-called ‘‘proton sponge’’ effect allowing endosomal escape [2,8,9] and transfer of pDNA to the nucleus [2,10]. Accordingly, PEI is effective in gene delivery into a variety of cell types even without the addition of endosomolytic agents [2,3]. Therefore, it is an ideal candidate molecule for the design of improved gene delivery systems. Polyethyleneglycol (PEG) modification (PEGgrafting) often can improve the solubility of macromolecules, minimize aggregation of particles and reduce their interaction with proteins in physiological fluids [11–15]. In spite of the advantages of PEI, the uptake into cells occurs by adsorptive endocytosis and, thus, is unspecific. Therefore, more specific methods of polyplex targeting are required for in vivo applications where gene delivery to defined target tissues is clearly desirable. Such targeting can be achieved by receptor-mediated gene delivery, i.e. coupling of ligands to the gene carrier to induce specific receptor binding and endocytosis. Moreover, an advantage of gene delivery technique using receptor-mediated endocytosis is prolonged duration of gene expression of targeted cells [16–18]. Therefore, various modifications of PEI have been explored in recent years and different strategies for targeted gene transfer using PEI and peptides, proteins, carbohydrates or glycoproteins have been recently investigated and reviewed [19]. Receptor-mediated transfer of galactosylated PEI–pDNA complexes into hepatocytes via asialoglycoprotein receptor [20–22], lactosylated PEI–pDNA complexes into airway epithelial cells [23] and mannosylated PEI–pDNA complexes into macrophages and dentritic cells via the mannose receptor [24] has been described. Based on these observations, we focused on using uronic acids, which are structurally related to carbohydrates, as novel targeting ligands for receptor-mediated gene delivery. In order to combine the useful properties of PEI and PEI–PEG with ligand-specific targeting, we synthesized trifunctional conjugates comprising (i) a nucleic acid binding domain, i.e. PEI, (ii) shielding properties, i.e. PEG-grafting, and (iii) a targeting moiety, i.e. uronic acids. We investigated the changes of transfection when gene vectors were functionalized with uronic acids. The basic polymers for the vectors were PEI and PEI–PEG. Uronic acids were directly bound to the polymers and via a PEG spacer. We expected that the transfection efficiency on human hepatocellular carcinoma cells (HepG2), human cervix carcinoma cells (HeLa), and human bronchial epithelial cells (16HBE14o
) would increase if the uronic acids bind to cell receptors. Biophysical values such as size and zeta potential were investigated as a function of the chemical structure of the polymers (Fig. 1).

O

OH

OH OH O

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O

OH OH O

OH OH

galacturonic acid(GalAc)

OH OH

OH mannuronic acid (ManAc)

OH

OH CH2 OH O OH

OH

OH H H

O

OH

OH

O

OH lactobionic acid(LacAc)

Fig. 1. Chemical structures of uronic acids used for grafting to PEI and PEI–PEG are galacturonic acid (GalAc), mannuronic acid (ManAc) and lactobionic acid (LacAc).

polyethyleneglycol8 monomethyl ether (M w ¼ 350 g=mol), O,O0 -bis(3aminopropyl)-polyethyleneglykol32 (M w ¼ 1500 g=mol) and all other chemicals were obtained from Sigma-Aldrich. The PEI is highly branched with a molar ratio of primary to secondary to tertiary amino groups of 1:2:1 and has a molar mass of M w ¼ 25 000 g=mol [information from the supplier (BASF)]. After its purification and removal of lower molar mass fractions by ultrafiltration (modified PES membrane, open channel, cutoff 10,000 g/mol, 12 h) the molar mass characteristics were M AUZ ¼ w 36 000 g=mol and M w =M n ¼ 1:5. All other chemicals were used without further purification. The plasmid pCMV-Luc containing the Photinus pyralis luciferase pDNA under the control of the cytomegalovirus (CMV) promoter was kindly provided by E. Wagner (Department of Pharmacy, Ludwig Maximilians University, Munich, Germany). The plasmid was propagated in Escherichia coli and purified by PlasmidFactory GmbH & Co. KG (Bielefeld, Germany). The purity (LPS) of this plasmid is p0.1 E.U./mg DNA, the amount of supercoiled pDNAX90% ccc.

2.2. Synthesis 2.2.1. Nitroxide-mediated synthesis of mannuronic acid (ManAc) by selective oxidation of primary alcohols ManAc was synthesized as described by de Nooy et al. [25]. Briefly, methyl-a-D-mannopyranoside (10.0 g, 0.0515 mol) as well as tetramethylpiperidin-1-oxyl-radical (TEMPO, 0.0523 g, 3.35  10
4 mol) and sodium bromide (1.033 g, 10.04 mol) were dissolved in water (100 mL). A further solution of sodium hypochlorite solution (77.25 mL with a concentration of 14% (w/w), 0.1545 mol) was adjusted to pH 10 with hydrochloric acid (1 M). Both solutions were cooled in an ice bath to 0–2 1C. Subsequently, the sodium hypochlorite solution was given to the solution containing the methyl-a-D-mannopyranoside. The pH value was controlled and adjusted constantly to pH 10 with 1 M sodium hydroxide. After 2 h the reaction was stopped by adding 13 mL of ethanol. The mixture was adjusted with hydrochloric acid (1 M) to pH 7 and freeze dried. For purification, the resulting raw product was dissolved in methanol (100 mL), precipitated salt was filtered off and the solvent was distilled. This procedure was repeated three times to provide ManAc (8.1 g, 194.1 mol) in 76% yield. 1H NMR (D2O): d (ppm) ¼ 3.5–3.8 (broad multiplet of the protons at C2–C4 of the sugar ring), 3.3 (doublet, O–CH3), 4.6 (proton at C5 of the sugar ring). 2.2.2. Cleaving of the protecting group at the glycosidic linkage Methyl ManAc was dissolved in hydrochloric acid (75 mL, 1 M) and heated to 95 1C for 20 min. After cooling to room temperature, the reaction was stopped with sodium hydroxide solution (75 mL, 1 M) and the water was removed in vacuum. Sodium chloride is a by-product of the reaction, which does not disturb the grafting of the ManAc to PEI. The unshielded ManAc was therefore used without further purification.

2. Experimental procedures 2.1. Materials Galacturonic acid (GalAc) and lactobionic acid (LacAc) were purchased from Fluka. Methyl-a-D-mannopyranoside was purchased from Fluka and modified as described in the Synthesis section. Methoxy

2.2.3. Synthesis of uronic acids functionalized PEI–PEG The PEI-graft–PEG was synthesized as described by Sedla´k and coworkers [26] in two steps: 1. Methoxy polyethyleneglycol glycidyl ether: Polethylenglycol monomethyl ether (35.0 g, 0.1 mol) was added to a mixture of epichlorhydrin

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(27.8 g, 0.3 mol), sodium hydroxide (3.2 g, 0.08 mol) and water (0.5 mL) and heated to 60 1C for 16 h and additionally to 90 1C for 1.5 h in an argon atmosphere. The product was dissolved in dichloromethane (150 mL) and water (100 mL) was added. Solid NaH2PO4 was used to adjust the pH of the water phase to 7. After phase separation, the organic phase was washed twice with 50 mL of water. The product was obtained as a yellow solution after distillation with a yield of 91%. Impurities of epichlorhydrin were identified via chloride test with silver nitrate. 1H NMR (D2O): d (ppm) ¼ 3.5–3.7 (broad multiplet, CH2–O, 32H), 3.4 (singulet, O–CH3, 3H), 2.95 (triplet, CH, 1H), d ¼ 2:78 (triplet, CH2, 2H) 2. PEI-graft–PEG. A solution of PEI (20.0 g, 0.56 mmol) dissolved in methanol (50 mL) was added to a solution of methoxy polyethyleneglycol glycidyl ether (6.0 g, 14.8 mmol) in methanol (20 mL). The homogeneous mixture was heated to 80 1C for 16 h. The mixture was purified by dialysis against distilled water using a membrane with a cut-off of 3500 g/mol to remove uncoupled PEG and the methanol. After final lyophilization, the product was obtained as a waxy yellow gel with a yield of 75%. 1H NMR (D2O): d (ppm) ¼ 2.4–2.8 (broad multiplet, CH2–N), 3.5–3.7 (broad multiplet, CH2–O). 2.2.4. Grafting of uronic acids to PEI and PEI–PEG ManAc (0.106 g, 0.5 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.105 g, 0.55 mmol) were dissolved in 10 mL water. Subsequently, N-hydroxysuccinimide (NHS) (0.063 g, 0.55 mmol) was added to the solution. After 3 min, a solution of O,O0 -bis-(3-aminopropyl)polyethyleneglycol (0.750 g, 0.5 mmol) in water (5 mL) was added. After 2 h, additional 0.105 g EDC, 0.063 g NHS in water (5 mL) and an aqueous solution (5 mL) of succinic acid (0.059 g, 0.5 mmol) were given to the mixture. After further 2 h, an amount of 0.105 g EDC and 0.063 g NHS dissolved in 5 mL of water were added. Finally a solution of PEI (1.8 g, 0.05 mmol) in 10 mL water was added. The mixture was stirred under an argon gas flow at room temperature over night. Then the resulting raw product was placed into a dialysis tube (cut-off 3500 g/mol) for a week. The product ManAc–PEI– PEG was obtained as a yellow solid after lyophilization with a yield of 35%. All other uronic acid copolymers with and without PEG side chains were synthesized in the same way. Concentration of each of the polymer solutions were determined according to the method of Ungaro [27] based on a copper sulfate assay and stock solutions of 2 mg/mL were prepared. 2.2.5. Synthesis of dextran conjugates ManAc (4.6 mg, 0.021 mmol) and EDC (4.6 mg, 0.024 mmol) were dissolved in 10 mL water. Subsequently, NHS (2.7 mg, 0.024 mmol) was added to the solution. After 3 min a solution of O; O0 -bis-(3-aminopropyl)polyethyleneglycol (32.0 mg, 0.021 mmol) in water (10 mL) was added. After 2 h, additional 4.6 mg EDC, 2.7 mg NHS in water (5 mL) and an aqueous solution (5 mL) of succinic acid (2.5 mg, 0.021 mmol) were given to the mixture. After further 2 h, an amount of 4.6 mg EDC and 2.7 mg NHS dissolved in 5 mL of water were added. Finally a solution of amino dextran (0.15 g, 0.0021 mmol) in 10 mL water was added. The mixture was stirred under an argon gas flow at room temperature over night. Then the solution were adjusted at pH 7 and the mixture were cooled to 0 1C. Carboxy-fluorescein-N-succinimidyl ester (FLUOS, 1.0 mg, 0.0021 mmol) were added as a solid. After stirring for 2 h at 0 1C the resulting raw product was placed into a dialysis tube (cut-off 3500 g/ mol) for a week in the fridge. The product was obtained as a yellow solid after lyophilization. The amount of ManAc per dextran were determined by UV/VIS. 2.2.6. Analytical methods The 1H-NMR spectra were measured with a DPX 400 Bruker spectrometer (Germany) at 400 MHz and 25 1C in deuterium oxide. The chemical shifts were referenced to the signal of deuterium oxide (dð1HÞ ¼ 4:72 ppm, DHO).

Size exclusion chromatography (SEC) was performed using an eluent of aqueous 0.2 M Na2SO4 solution with 1% acetic acid. The SEC was equipped with a UV detector (Thermofinningen, Germany), a refractive index detector (Wyatt Technology Deutschland GmbH, Germany), a viscosity detection system (WGE Dr. Bures, Germany) as well as a multiangle laser light scattering detector (Wyatt Technology Deutschland GmbH, Germany). The SEC columns used were fife TSK columns in a row (Tosohaas, Japan): first a TSK gel PWH guard column (7.5  75 mm, 12 mm particle size), second a TSK gel 66,000 PW (7.5  300 mm, 17 mm particle size), third a TSK gel 65,000 PW (7.5  300 mm, 17 mm particle size), fourth a TSK gel 64,000 PW (7.5  300 mm, 17 mm particle size), and the fifth a TSK gel 63,000 PW (7.5  300 mm, 10 mm particle size). Analytical ultracentrifugation (AUC) was carried out using an Optima XL-1 ultracentrifuge (Beckman-Coulter, USA). Rayleigh interference optics was used for detection. The samples were investigated in titanium double sector centerpieces (optical pathway 12 mm) at 50,000 rpm and 20 1C using 0.01 M NaCl solution as solvent. The relative molar mass Mr of the polymers was calculated as described by Linow and Philipp [28] with   s0  Z0 3=2 ð½Z  kSB Þ1=2 , M r ¼ 2:406  1025 1
v  r0 where s0 is the Svedberg constant in s, [Z] is the intrinsic viscosity of the Schulz–Blaschke equation in cm3/g, kSB is the constant of the Schulz– Blaschke equation, Z0 is the viscosity of the solvent (Z0 ¼ 0:01 mPa s), 1
v is the specific volume of the polymer and r0 is the density of the solvent in g/cm3. The specific volume of the polymer was determined with a DMA 60/602 density meter (Anton Paar, Austria). The measurement was performed in 0.01 M NaCl solution using five different polymer concentrations. The viscosity measurements were performed with an automatic dilution viscometer TI 1 (SemaTech, France). The intrinsic viscosity was determined in 0.01 M NaCl solution. The uronic acid concentration was determined in a Cary 100 spectrometer (Varian). Absorbance of uronic acid was measured by the dinitro salicylic acid assay [29] at a wavelength of 560 nm. This method is only applicable for sugars with a semi-acetal. The degree of substitution was calculated with the help of a calibration curve using five known uronic acid concentrations (0.01–1.0 mg/ml). The amount of LacAc per polymer was measured by the anthron [30] method at 630 nm. 2.2.7. Cell lines HeLa cells were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; DMSZ No.: ACC 57). The cell line 16HBE14o
derived from human respiratory epithelial cells was generously provided by Dieter C. Grunert (University of California at San Francisco, CA, USA). HepG2 cells were obtained from DSMZ (DSMZ No.: ACC 180). The cells were grown in 10% fetal calf serum (FCS) from Gibco-BRL (Karlsruhe, Germany) supplemented with minimum essential medium (MEM, Gibco-BRL) at 37 1C in a 5% CO2 humidified air atmosphere. 2.2.8. Preparation of gene vector complexes pDNA (pCMV-Luc), PEI and uronic acids functionalized PEI conjugates, respectively, were diluted separately in 25 mL of HEPES buffered saline (HBS) (150 mM NaCl, 10 mM HEPES, pH 7.3, sterile filtered through a 0.2 mm membrane filter). The pDNA solution (25 mL) was gently added to the PEI solution or the uronic acid–PEI-conjugate solutions (25 mL), respectively, and thoroughly mixed by pipetting. A total of 0.5 mg pDNA per well was complexed with PEI and uronic acidsfunctionalized PEI conjugates, respectively, at N/P ratios (molar ratio of PEI nitrogen to pDNA phosphate) of 1.25, 2.5, 5, 10, 20 and 40. The resulting gene vector complexes were incubated for 20 min at room temperature before use. 2.2.9. In vitro transfection procedure and luciferase activity measurement For transfection cells (15 000 cells per well, 96-well microplate) were seeded 1 day before transfection and grown in MEM containing 10%

ARTICLE IN PRESS S.I. Weiss et al. / Biomaterials 27 (2006) 2302–2312 FCS. At a confluency of about 80%, cells were washed with phosphatebuffered saline (PBS, Gibco-BRL) and then covered with 150 mL of medium in the absence of FCS. Then, 50 mL of gene vector complexes, corresponding to 0.5 mg of pDNA, were pipetted onto the cells. After 4 h of incubation at 5% CO2 and 37 1C, the medium was replaced with 10% FCS-containing medium supplemented with 0.1% (v/v) penicillin/streptomycin and 0.5% (v/v) gentamycin (Invitrogen). Twenty-four hours later luciferase activity was measured [31]. 2.2.10. Size and zeta potential measurement The particle sizes (volume average of hydrodynamic diameter) were determined by dynamic light scattering (DLS) and the zeta potentials were measured (ZetaPALS/Zeta Potential Analyzer; Brookhaven Instruments Corporation, Austria). Gene vector solutions in distilled water were generated at a pDNA concentration of 30 mg/ml. The following settings were used: ten sub-run measurements per sample; viscosity for water 0.89 cP; beam mode F ðKaÞ ¼ 1:50 (Smoluchowsky); temperature 25 1C. 2.2.11. Fluorescence microscopy The cells (105 cells/chamber, 8-chamber slides; Becton Dickinson Labware, Franklin Lakes, NJ. USA) were seeded 1 day before addition of FLUOS-dextran-conjugates and grown in MEM containing 10% FCS. The cells were washed with PBS (Gibco-BRL) and then 2 mM FLUOSlabelled conjugates diluted in MEM were added for 45 min (37 1C, 5% CO2). Some of the cells were incubated with a 11-fold molar excess of ManAc (50 mM) for 30 min. After washing the cells with PBS, the cells were rinsed, fixed in 4% paraformaldehyde for 10 min and afterwards nuclei were counterstained with 0.33 mM DAPI (40 ,6-diamidino-2-phenylindole). The dishes were covered with mounting medium (Vectashield, Vector Laboratories Inc., Burlingame, CA, USA). An epifluorescence Axiovert 135 microscope (Zeiss, Jena, Germany) was applied for microscopy. 2.2.12. FACS analysis The cells (105 cells/well; 24-well plates) were seeded 1 day before addition of FLUOS-dextran-conjugates and grown in MEM containing 10% FCS. The cells were washed with PBS (Gibco-BRL) and then 2 mM FLUOS-labelled conjugates diluted in MEM were added for 45 min (37 1C, 5% CO2). Some of the cells were incubated with a 11-fold molar excess of either ManAc or Mannose (50 mM) for 30 min. After washing the cells with PBS, the cells were trypsinized and analyzed by FACS (FACScan, Becton Dickinson). 2.2.13. Statistical analysis The results are reported as means7standard deviation. The statistical analyses between different groups were determined with a non-paired ttest. Probabilities of pp0:05 were considered as significant. All statistical analyses were performed using the StatView 5.0 program (SAS Institute Inc., Cary, NC, USA).

3. Results and discussion 3.1. Number of ligands per PEI or PEI–PEG The number of LacAc ligands grafted to the PEI or PEI–PEG was found to be in the range of 9–11 ligands per polymer chain, which was theoretically expected from the stoichiometry of the reaction mixture. In contrast, the PEI and PEI–PEGs functionalized with ManAc and GalAc exhibited a lower number of ligands when the same graft conditions were used (3–4 ManAc-groups and 5–7 GalAcgroups per PEI–PEG molecule). The lower number of ManAc and GalAc ligands compared to LacAc could probably be due to steric hindrance of the ManAc and GalAc. The carboxylic group of LacAc is located at the

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side chain with a relatively large distance to the sugar ring and could, therefore, apparently react faster with the PEI compared to the carboxylic acid groups of ManAc and GalAc. 3.2. Hydrodynamic diameter of the PEIs after grafting with ligands The hydrodynamic diameter dh and the polydispersity indexes (PDI) of the polymers before and after grafting were determined by DLS. The hydrodynamic diameters of all PEIs grafted with uronic acids were within the range of 9–13 nm, which was close to the value of the non-grafted PEI (10 nm). This was unexpected since their molar masses increased after grafting. We conclude therefore that the grafting of the ligands to PEI changed the conformation, i.e., compactness of the PEI. A similar trend has previously been reported by Park and Choi [32] who described that branched PEI becomes more compact with increasing molar mass even when non-grafted. The grafting of PEG to PEI resulted in an increase of the hydrodynamic radius from 10 nm (PEI) to 17 nm (PEI–PEG), which was expected for the grafting of 20 PEG chains per PEI chain. After grafting of the ligands to PEI–PEG, we found increased diameters only for GalAc(5)–PEI–PEG (26 nm) and LacAc(9)–PEI–PEG (22 nm) but not for ManAc(4)– PEI–PEG (14 nm). These differences in the changes of the diameter could result from different orientations of the sugar rings. However, the particle size of the polymers, whose ligands were bound via PEG side chains, exhibit smaller values. This phenomenon could be seen clearly for (GalAc–PEG)5–PEI–PEG and (LacAc–PEG)10–PEI– PEG which displayed no larger size (19 and 17 nm) than PEI–PEG (17 nm). This is probably due to the flexibility of the PEG side chains (Fig. 2). 3.3. Molar mass and molar mass distribution It can be seen in Table 1 that the molar masses of PEI copolymers increased with the increasing numbers of grafted groups. The analysis of the uronic acid copolymers by SEC showed an increasing polydispersity with increasing degree of conversion. This was particularly to note for the uronic acid grafted PEI–PEGs. We suggest that this was due to the inhomogeneous grafting of PEI with PEG as evidenced by broadening of the molar mass distribution of the copolymers (data not shown). Apparently, this led to a mixture of graft copolymer PEI–PEG as well as PEI and graft copolymer fractions with non-uniform chemical composition. In a further modification with the uronic acid the ligands again preferentially reacted with the highmolecular mass polymers. This could lead to a rise of the polydispersity, partly seen as a formation of a shoulder within the high-molecular molar mass range of the elution diagram of the SEC. The molar masses and polydispersity increased as expected with increasing degree of conversion.

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Furthermore, highly substituted products displayed an increasingly more compact structure in aqueous solution. 3.4. Gene transfer efficiency of the novel uronic acidfunctionalized PEI and PEI–PEGS The investigation of gene transfer efficiencies of uronic acids–PEI–pDNA complexes was performed on HeLa, 16HBE14o
, and Hep-G2 cells. Plasmid DNA encoding a luciferase gene, pCMV-Luc, was complexed with uronic acid PEI-conjugates or PEI at N/P ratios of 1.25, 2.5, 5, 10, 7 1

6

2

5 4 g (s)

3

4

3 2 1 0 0

1

2

3

4

5

6

Sedimentation Constant [ 10-13 s ]

Fig. 2. Sedimentation constant distributions of PEI (1), PEI grafted with uronic acid (2), PEI–PEG (3), and PEI–PEG grafted with uronic acid (4).

20 and 40. Transfection efficiencies of GalAc–PEI–pDNA complexes increased with increasing N/P ratios and reached highest transfection levels between N=P ¼ 5215 (Fig. 3). At higher N/P ratios (N=P415) gene expression declined. Transfection efficiencies were significantly up to 100-fold lower than for PEI–pDNA complexes especially at low N/P ratios (Fig. 3A). Curve shapes were similar on all tested cell lines. These results suggested that grafting of PEI in close proximity of the carbohydrate ring could apparently interfere with receptor binding. Therefore, LacAc was grafted to PEI comprising a carbohydrate ring which is structurally related to galactose, but additionally comprises a gluconic acid open-chain allowing coupling of PEI via a four carbon spacer arm. The transfection profile of LacAc–PEI–pDNA was similar to GalAc–PEI–pDNA complexes (Fig. 3B). In order to test if the degree of targeting ligand modification of PEI (LacAc(9)–PEI) was not adequate to achieve receptor-mediated gene delivery, LacAc-functionalized polymers containing a two-fold higher degree of LacAc-modification (LacAc(20)–PEI) were analyzed. Transfection rates mediated by LacAc(20)– PEI were at the same level or lower than for LacAc(9)–PEI. These results suggest that neither GalAc nor LacAc are suitable targeting ligands for receptor-mediated gene delivery. In contrast to these observations, ManAc–PEI–pDNA and (ManAc–PEG)–PEI–pDNA complexes mediated significantly up to several magnitudes higher gene transfer efficiencies than the tested LacAc–PEI–, GalAc–PEIconjugates, and PEI at low N/P ratios (Fig. 3C). Whereas transfection rates increased with increasing N/P ratios for PEI–, GalAc– and LacAc–PEI–pDNA complexes

Table 1 Molecular characteristics of the grafted PEIs dh (nm)

PDI

MAUZ (103 g/mol) p

3 MSEC w,UC (10 g/mol)

M w =M n

3 MSEC w,LS (10 g/mol)

10 11 10 9 10 10 11 13

0.25 0.30 0.31 0.34 0.36 0.36 0.43 0.37

35.8 35.9 43.4 36.7 40.8 40.3 30.5 41.2

50.1 56.6 88.5 38.9 133.9 195.4 91.6 59.0

1.5 2.3 2.4 1.9 2.1 2.2 2.1 2.0

49.8 70.4 88.1 34.4 105.9 81.8 86.1 66.7

Poly(ethylenimine)s-graft–poly(ethylenglycol)s PEI–PEG(20) 17 GalAc(5)–PEI–PEG 26 (GalAc-PEG)5–PEI–PEG 19 LacAc(9)–PEI–PEG 22 (LacAc–PEG)10–PEI–PEG 17 ManAc(4)–PEI–PEG 14 (ManAcPEG)3 –PEI–PEG 15

0.40 0.42 0.41 0.44 0.44 0.38 0.43

56.2 61.0 59.3 60.4 55.6 44.2 48.6

74.0 304.1 212.5 195.4 220.0 135.7 146.8

1.9 1.9 3.2 3.4 2.7 3.2 3.2

102.7 344.5 200.0 154.6 145.5 109.9 140.7

Poly(ethylenimine)s PEI GalAc(7)–PEI (GalAc–PEG)5–PEI LacAc(9)–PEI LacAc(20)–PEI (LacAc–PEG)11–PEI ManAc(4)–PEI (ManAc–PEG)3–PEI

The number in brackets represents the average number of uronic acid groups per polymer molecule, dh is the hydrodynamic diameter, PDI is the is the molar mass calculated from the peak maximum of a sedimentation coefficient distribution, MSEC polydispersity index determined by DLS, MAUZ p w,UC is the molar mass using SEC with an universal calibration with PVP standards, DSEC is the polydispersity (M w =M n ) of the polymer resulting from SEC, MSEC W,LS is the molar mass using a MALLS (multi-angle laser light scattering) detector.

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(a maximum gene expression was between N=P ¼ 5 and 15), ManAc-functionalized PEI conjugates showed the highest gene expression at low N/P ratios with a slight decay at higher N/P ratios. This observation suggested that

Fig. 3. Investigation of gene transfer efficiency of uronic acids-functionalized PEI and PEI–PEG performed on HEP-G2, 16HBE14o
and HeLa cells. Plasmid DNA encoding a luciferase gene (pCMV-Luc) was complexed with PEI, PEI–PEG and GalAc–PEI-conjugates (A), LacAc– PEI-conjugates (B) and ManAc–PEI-conjugates (C) at N/P ratios of 1.25, 2.5, 5, 10, 20 and 40. Transfection was performed with 0.5 mg/well pDNA. The results of three independent experiments performed in quadruplicates are shown.

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the uptake-mechanism of ManAc–PEI-conjugates occurred apparently independently from unspecific charge interactions of the polyplexes with the cellular surface, which is commonly observed for cationic polymer-based gene delivery systems [33], but specifically through the ManAc ligand. Coupling of ManAc to PEI of the PEI–PEG polymer (ManAc(4)–PEI–PEG–pDNA) resulted only in a moderate increase of gene expression at low N/P ratios. Further, coupling of additional PEG-chains to (ManAc–PEG)–PEI ((ManAc–PEG)3–PEI–PEG) resulted

Fig. 3. (Continued)

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those of PEI directly coupled to GalAc (data not shown), indicating that superior transfection properties were caused by the ManAc ligand. GalAc differs from ManAc in the conformational orientation of two hydroxylic groups in positions 2 and 4 of the carbohydrate ring. The small difference in the chemical structures has obviously a strong influence on transfection efficiency. 3.5. The uptake of mannuronic acids functionalized PEI and PEI–PEG into cells is mediated by receptors To further proof that ManAc induced receptor-mediated gene delivery, the sizes and surface charges (zeta potentials) were analyzed. It has previously been reported that PEImediated gene delivery largely depends on the surface charge of the complexes. Positively charged complexes 50

PEI PEI-PEG

40

ManAc (4)-PEI (ManAc-PEG) 3-PEI

zeta potential in mV

30 20 10 0

0

2

4

6

8

10

12

-10 -20

(A)

N/ P-ratio

-30

PEI

350

PEI-PEG ManAc (4)-PEI

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(ManAc-PEG) 3-PEI

size in nm

250

Fig. 3. (Continued)

in lower gene transfer efficiencies compared to (ManAc– PEG)3-PEI. This was probably due to burying of the ManAc moiety through the additional PEG side chains resulting in a shielding effect which inhibited interaction of ManAc ligands with their receptors on the cellular surface. To test whether differences in the chemical synthesis could be responsible for higher transfection efficiencies of the ManAc–PEI–conjugates, we also synthesized GalAc by selective oxidation of methyl-a-D-galactopyranoside before grafting it to PEI and PEI–PEG. Transfection efficiencies of the resulting GalAc–PEI-conjugates were comparable to

200 150 100 50 0 0

(B)

2

4

6

8

10

12

N/P-ratio

Fig. 4. Characterization of PEI–pDNA–, PEI–PEG–pDNA–, ManAc(4)– PEI–pDNA-, and (ManAc–PEG)3–PEI–pDNA-complexes. Gene vector solutions in distilled water were generated at a pDNA concentration of 30 mg/mL. Zeta potentials were measured and particle sizes were determined by dynamic light scattering.

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were required for interaction with the negatively charged cell surface to mediate efficient gene delivery. Zeta potentials of the complexes were negative at N=P ¼ 1:25 and positively charged at N=P ¼ 5 and 10 independent of the polymer structure (Fig. 4A). The size of the complexes decreased with increasing N/P ratios independent of the polymer structure (Fig. 4B). Both ManAc modification and PEG-grafting induced an increase of particle sizes by 2–3fold. Further, all of the particles showed broad size distributions (PDI40:2, data not shown). These results demonstrated that the high transfection rates of ManAc– PEI–pDNA complexes at low N/P ratios were presumably mediated by the ManAc moiety, as negatively charged gene vectors without receptor binding should not efficiently bind to the negatively charged cellular surface and, thus, result in only low transfection levels. Indeed, negatively charged unmodified PEI–pDNA complexes mediated only low transfection levels. Further, important for high transfection efficiency of ManAc–PEI–conjugates was the cleavage of the protecting group at the glycosidic linkage before transfection. Methyl-a-D-ManAc mediated only low transfection rates (data not shown). To further proof that ManAc modification resulted in receptor-mediated gene delivery, transfection experiments were performed in the

presence of excess of ManAc. As shown in Fig. 5, gene expression significantly decreased in the presence of a twenty-five-fold molar excess of ManAc for only ManAc– PEI– and –PEI–PEG on each of the cell lines tested. These results demonstrated that ManAc modification resulted in receptor-mediated gene delivery. 3.6. Mannuronic acid-mediated cellular uptake of fluorescently labelled dextran conjugates To further investigate ManAc-mediated cellular uptake, cells were incubated with either fluorescence labelled dextran (FLUOS-Dex) and ManAc-modified dextran (FLUOS-ManAc–Dex) or PEG–dextran-graft-copolymers (FLUOS-PEG–Dex) and (ManAc–PEG)–dextran-graftcopolymers (FLUOS-(ManAc–PEG)–Dex) both in the absence and presence of a 25-fold molar excess of ManAc. Only negligible cellular uptake of FLUOS-Dex, FLUOSPEG–Dex and FLUOS-ManAc–Dex and FLUOS-(ManAc–PEG)–Dex in the presence of excess of ManAc was observed independent of the cell line tested (Fig. 6, only shown for 16HBE14o- cells). In contrast, strong spot-like cellular fluorescence was observed for ManAc-conjugated dextrans on each of the cell lines tested. The spot-like

16HBE14o-

5.0E+06

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HEP-G2

2.0E+05

- mannuronic acid

- mannuronic acid + mannuronic acid

RLU / 10s / mg protein

3.0E+06

2.0E+06

1.5E+05

+ mannuronic acid

1.0E+05

5.0E+04

1.0E+06

*

*

*

0.0E+00

*

*

G )3 -P EI ( 4) (M an PE Ac I- P -P EG EG )3 -P EI -P EG PE I-P EG (2 0) M an

Ac

-P E

Ac (4 )-P EI (M

HELA

1.5E+07

RLU / 10s / mg protein

an Ac

M an

PE I

G )3 -P EI Ac (4 (M )-P an E Ac I- P -P EG EG )3 -P EI -P EG PE I-P EG (2 0) M an

an Ac -P E

Ac (4 )- P EI

0.0E+00

(M

M an

*

*

*

PE I

RLU / 10s / mg protein

4.0E+06

1.0E+07

- mannuronic acid + mannuronic acid

5.0E+06

*

*

*

* PE I

G )3 -P EI (4 )-P an EI Ac -P -P EG EG )3 -P EI -P EG PE I-P EG (2 0) (M

M an

Ac

-P E an Ac

(M

M an

Ac

(4 )-P EI

0.0E+00

Fig. 5. Investigation of the uptake-mechanism of ManAc-functionalized PEI-conjugate–pDNA complexes on HEP-G2, 16HBE14o- and HELA cells. Before transfection, cells were incubated with a twenty-five-fold molar excess of ManAc. Plasmid DNA encoding a luciferase gene (pCMV-Luc) was complexed with PEI, PEI–PEG and ManAc–PEI-conjugates at an N/P ratio of 1.25. Transfection was performed with 0.5 mg/well pDNA. Significant differences are indicated by  po0:05. The results of three independent experiments performed in quadruplicates are shown.

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fluorescence pattern strongly suggested internalization of the FLUOS-ManAc–Dex conjugates via the endosomal pathway. Furthermore, we investigated ManAc-mediated cellular uptake via FACS analysis. 16HBE14o
cells were incubated with FLUOS-PEG–Dex and FLUOS-(ManAc– PEG)–Dex both in the absence and presence of a 25fold molar excess of either ManAc or mannose. Cellular uptake of FLUOS-PEG–Dex and FLUOS-(ManAc– PEG)–Dex in the presence of excess of ManAc was negligible whereas uptake of FLUOS-(ManAc-PEG)Dex in the presence of excess of mannose was not inhibited (Fig. 7, only shown for 16HBE14o- cells). These observations indicate the presence of ManAc-receptors on the cell

surface which allowed specific cellular uptake of conjugated polymers. 4. Conclusion In conclusion, we synthesized well-defined uronic acidand gluconic acid-functionalized PEI and PEI–PEG as novel synthetic gene delivery carriers. We could demonstrate in this study that the transfection efficiency was dependent on (i) PEG-grafting, (ii) type of uronic acid used for modification, and (iii) its degree of modification. In contrast to GalAc and LacAc, ManAc represents a novel highly efficient targeting ligand ideal for receptor-mediated

Fig. 6. Analysis of mannuronic acid-mediated cellular uptake of fluorescently labelled dextran conjugates on 16HBE14o- cells by fluorescence microscopy. Cells were incubated with 2 mM fluorescently labelled dextran (FLUOS-Dex), PEG–dextran-block copolymers (FLUOS-PEG–Dex), ManAc modified dextran (FLUOS-ManAc–Dex) and (ManAc–PEG)–dextran block copolymers (FLUOS-(ManAc–PEG)–Dex) diluted in MEM for 45 min (5% CO2; 37 1C). Cells treated with FLUOS-ManAc–Dex and FLUOS-(ManAc–PEG)–Dex were additionally incubated in the presence of a twenty-five-fold molar excess of ManAc. FLUOS-Dex (a), FLUOS-PEG–Dex (b), FLUOS-ManAc–Dex (c), FLUOS-(ManAc–PEG)–Dex (d), and FLUOS-ManAc–Dex (e), FLUOS-(ManAc–PEG)–Dex (f) additionally incubated in the presence of a 25-fold molar excess of ManAc.

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40

Counts

30 M1 20

10 M2 0 100

(A)

101

102

103

104

FLUOS

40

Counts

30 M1 20

10 M2 0 100 (B)

101

102

103

104

FLUOS

Fig. 7. Analysis of mannuronic acid-mediated cellular uptake of fluorescently labelled dextran conjugates on 16HBE14o- cells by FACS. Cells were incubated with 2 mM FLUOS-PEG–Dex (A) and FLUOS(ManAc–PEG)–Dex (B) diluted in MEM for 45 min (5% CO2; 37 1C). Cells treated with FLUOS-(ManAc–PEG)–Dex were additionally incubated in the presence of a 25-fold molar excess of either ManAc or mannose. Violet: 16HBE14o- cells untreated, yellow: FLUOS-PEG–Dex (A) or FLUOS-(ManAc–PEG)–Dex (B) in the absence of ManAc or mannose, red: FLUOS-(ManAc–PEG)–Dex in the presence of ManAc, green: FLUOS-(ManAc–PEG–)Dex in the presence of mannose.

gene delivery. Therefore, ManAc-modification of synthetic gene carriers could represent a novel tool to increase gene delivery efficiency on various cell types which could be promising for in vivo applications. Acknowledgments This work was supported by the 5th framework program of the European Union (Grant no. QLK3 CT 2002 02119), the Max Planck Society, the Fraunhofer Society, the Federal Institute for Materials Research and Testing, and in parts by the BMBF BioFuture program (FKZ 0311898). References [1] Marshall E. Gene therapy. Second child in French trial is found to have leukemia. Science 2003;299:320.

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