Substrate-mediated DNA delivery: role of the cationic polymer structure and extent of modification

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Substrate-mediated DNA delivery: role of the cationic polymer structure and extent of modification Tatiana Segura a, Matthew J. Volk a, Lonnie D. Shea a,b,* a b

Department of Chemical Engineering, Northwestern University, 2145 Sheridan Road E156, Evanston, IL 60208-3120, USA Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road E156, Evanston, IL 60208-3120, USA Received 19 March 2003; accepted 9 August 2003

Abstract DNA complex immobilization to substrates that support cell adhesion can enhance gene transfer by maintaining DNA within the cellular environment while limiting complex aggregation. This report examines the tether design (e.g., extent of functionalization) and cationic polymer structure for their effect on complex binding to the substrate and cellular transfection. DNA is complexed with cationic polymers (polylysine, PL; polyethylenimine, PEI), which are functionalized with biotin for binding to a neutravidin (NA) substrate. Surfaces densities ranging from 0.4 to 2.6 Ag DNA/cm2 were obtained for PL, and from 0.7 to 1.0 Ag DNA/cm2 for PEI. The distribution of biotin groups for PL/DNA complexes had a dual effect on cellular transfection. Increasing the fraction of PL with biotin residues decreased luciferase activity; however, increasing the number of biotin residues per PL increased luciferase activity. For PEI, the number of biotin groups present on the complex did not affect transgene expression. Release studies demonstrated that 20 – 30% of the immobilized DNA was released over 8 days, with 8 – 20% released during the first 24 h. Enzymatic degradation of cationic polymers is not necessary for transfection. Additionally, the duration of transgene expression was extended for surface-mediated delivery relative to bolus delivery. D 2003 Elsevier B.V. All rights reserved. Keywords: Polylysine; Polyethylenimine; Vector unpacking; DNA immobilization; Solid-phase delivery

1. Introduction Controlled and efficient gene delivery has implications to many fields ranging from basic science to clinical medicine. Current strategies to enhance nonviral gene delivery involve the complexation of DNA with cationic polymers or lipids, and polymeric delivery. Cationic polymers or lipids can self-assemble * Corresponding author. Department of Chemical Engineering, Northwestern University, 2145 Sheridan Road E156, Evanston, IL 60208-3120, USA. Tel.: +1-847-491-7043; fax: +1-847-491-3728. E-mail address: [email protected] (L.D. Shea). 0168-3659/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2003.08.003

with DNA to form particles that are capable of being endocytosed by cells [1]. Complexation functions to reduce the surface charge density, effective size, and cellular degradation of DNA [2]. These complexes are often delivered as a bolus, such as addition to culture wells in vitro or injection into a tissue in vivo. Bolus delivery of these complexes can be limited by mass transport limitations or deactivation processes, such as degradation, aggregation, or clearance from the tissue. For example, in vitro studies have estimated that bolus addition of complexes to the culture media results in internalization of approximately 20 –50% of the DNA added [3]. These limitations have provided the moti-

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vation for the development of alternative delivery strategies. The controlled delivery of DNA complexes from biomaterials offers the potential to enhance gene transfer by maintaining an elevated concentration of DNA within the cellular microenvironment [4]. Increasing the concentration of DNA in the cellular microenvironment by adsorbing DNA to silica particles that settle on the surface increased transfection [5]. DNA delivery from biomaterials can be categorized into two fundamental approaches: sustained release and immobilization. Sustained-release systems are designed to maintain elevated concentrations locally by supplying DNA to balance the loss by degradation or clearance. The continued presence of the plasmid during cell division may also facilitate entry into the nucleus, during which the nuclear membrane is compromised [6]. Alternatively, DNA can be immobilized within or to a biomaterial scaffold. Gene transfer using collagen is hypothesized to function by maintaining the DNA in situ, possibly due to limited transport through the collagen, until internalization by cells present locally [7,8]. A similar strategy has been used by some viruses, which associate with extracellular matrix molecules (e.g., fibronectin) for enhanced uptake [9,10]. More recently, synthetic systems that specifically bind viruses [11,12] or nonviral DNA complexes [13] to a polymeric substrate are being developed. Immobilization of the DNA to the substrate to which cells adhere maintains the DNA in the cell microenvironment for subsequent cellular internalization. Synthetic systems based on the immobilization of nonviral DNA complexes have a guiding principle that the substrate must be designed to maintain the DNA locally, yet allow for cellular internalization. In this report, we have examined the morphology, substrate binding, release profile, and transgene expression for cationic polymer/DNA complexes that are tethered to a cell-adhesive substrate. Binding and transfection were characterized for cationic polymers that are primarily composed of primary amines (polylysine, PL) or secondary amines (linear polyethylenimine, PEI). DNA was immobilized by modifying the cationic polymer (PL or PEI) with biotin for binding to a neutravidin (NA)-modified substrate. A fraction of the condensing cationic polymers was covalently modified with biotin residues (Fig. 1). The complexes

Fig. 1. Schematic of the substrate-mediated delivery strategy. CP represents the cationic polymer used for condensation.

were then tethered to substrates capable of binding biotin. The extent of cationic polymer modification with biotin and the quantity of modified polymers per complex were characterized for their influence on substrate binding, release from the substrate, and cellular transfection. Finally, the duration of gene expression resulting from substrate-mediated delivery was examined.

2. Experimental 2.1. Materials Plasmid DNA encoding for luciferase (pNGVL1Luc) was purified from bacteria culture using Qiagen (Santa Clara, CA) reagents and stored in Tris – EDTA buffer solution (pH 7.4). Three PL peptides were used for DNA complexation: Cys – Trp –Lys19 (K19; BioPeptide, San Diego, CA), LLys214 (K214, average molecular weight of 27,400; Sigma, St Louis, MO), and DLys214 (DK214, average molecular weight of 27,200; Sigma). Note that the subscript represents the average degree of polymerization for the peptide.

Linear PEI and biotinylated PEI (PEI– biotin) were purchased (7 mM amine content; PolyTransfection, Strasbourg, France) and manufacturer specifications indicate that the extent of biotin modification is in the range of 7 – 10 mol biotin/mol PEI. Neutravidin and biotin reagents for peptide modification and surface tethering were purchased from Pierce (Rockford, IL). All other reagents were obtained from Fisher Scientific (Fairlawn, NJ) unless otherwise noted. 2.2. Synthesis of biotinylated polylysine The PL peptides (K214, DK214) were biotinylated using succinimide ester (NHS)/amine chemistry, as described previously [13]. Briefly, K214 or DK214 [10 mg in 1 ml of phosphate-buffered saline (PBS), pH 7] was mixed with EZ-link-Sulfo-NHS-LC-Biotin (2.8 mg) and incubated for 2 h at 4 jC. The reaction mixtures were purified using dialysis cassettes and a monomeric avidin column to separate the biotinylated components from the nonbiotinylated species. The purified biotinylated peptides (K214-B) were then lyophilized and stored as powder at 20 jC. The degree of biotinylation of the PL peptides was determined by quantifying the mole ratio of biotin to K214 using 2[4V-hydroxyazobenzene]-benzoic acid (HABA assay; Pierce). 2.3. Radiolabeling of plasmid DNA Plasmid DNA was radiolabeled with a a-32P dATP using a nick translation kit (Amersham Pharmacia Biotech, Piscataway, NJ). The labeling reaction was performed following the manufacturer’s protocol with minor modifications. DNA was diluted to 100 ng/Al (2 Ag in 20 Al) in distilled water and mixed with 20 Al of a dCTP/dGTP/dTTP solution (7 Al of each, 300 AM) and an enzyme solution (20 Al) composed of DNA polymerase I (0.5 U/Al) and DNase I (10 pg/Al). The radiolabeled nucleotide a-32P dATP (250 ACi/20 Al) was added last (20 Al) and the solution was mixed gently by pipetting. The reaction was incubated for 2 h at 16 jC. To stop the reaction, 0.2 M EDTA solution was added (10 Al, pH 8) and the solution was heated to 97 jC for 5 min. The reaction mixture was purified using QIAquick spin column (Qiagen). The resulting pure 32P-labeled

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DNA (32P DNA, 0.04 Ag/Al) was further diluted with unlabeled DNA to produce a 1% solution of labeled plasmid. 2.4. Complex formation and surface tethering The ability of the PL peptides (K214-B and DK214-B) and PEI (PEI, PEI – biotin) to condense DNA was assessed by gel electrophoresis. The cationic polymers were mixed and added in a stepwise manner to a DNA solution (300 Al of 20 Ag/ml). After each addition step, the solution was vortexed, incubated for 5 min, and a sample removed. Trypsin/EDTA solution was added to the PL complexes containing the highest charge ratio ( F ) and incubated at 37 jC for 1 h. Gel electrophoresis was performed to assess the extent of complex formation for the samples. DNA complexes were incubated on neutravidin surfaces for specific tethering through the biotin –NA interaction. For PL complexation, DNA (60 Al of 66.7 Ag/ml) was complexed with PL at charge ratios ( F ) ranging from 0.6 to 5.5. Prior to mixing, the PLs were diluted to a final volume of 40 Al with Tris-buffered saline (TBS; 100 mM Tris and 150 mM NaCl, pH 7.4). DNA (50 Al of 40 Ag/ml) diluted in 150 mM NaCl, as recommended by the manufacturer, was complexed with PEI at a nitrogen/phosphate ratio (N/P) of 5. Prior to mixing, the PEI solution was diluted to a final volume of 50 Al with 150 mM NaCl. The solution containing the cationic polymers was then added to the DNA solution. The number of tethers on each complex was varied by mixing biotinylated and nonbiotinylated peptides prior to complexation with DNA. After mixing, the solution was transferred to NA surfaces and incubated for 2 h at room temperature. The unbound complexes were then removed and the surfaces were washed with TBS. Surface binding of the complexes was determined by either removal of the complexes and measurement with either a fluorescent dye or 32P DNA. For PL, the surfaces were incubated with trypsin (100 Al) at 37 jC for at least 2 h to degrade the PL and to release the DNA into the solution. The quantity of DNA was measured with a fluorometer (Turner Designs TD-360) using the Hoechst dye. The surface density of DNA/PEI complexes was determined using 32P-labeled DNA. DNA/PEI complexes were

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formed using 32P DNA and immobilized to the surface using the procedure described above. Following immobilization of the DNA/PEI complexes, individual strip plate wells were cut and placed in scintillation cocktail (5 ml; ScintiVerse II) and read in a scintillation counter. The amount of radioactivity observed was correlated to DNA mass with a standard curve. Complex formation was visualized using a fluorescein-tagged h-galactosidase vector (Gene Therapy Systems, San Diego, CA). Complexes were made with 100% K214 or K214-B and incubated on NA surfaces. Images of the complexes were captured using a Leica fluorescence microscope equipped with a digital camera. 2.5. DNA/cationic polymer complex release The percentage of DNA/cationic polymer complexes released from the surface was determined using 32P DNA complexes immobilized to NA plates. Following the washing procedure, 160 Al of phosphate buffer saline (pH 7.4) was added to each well and the surfaces were incubated at 37 jC in a humid chamber. At predetermined time points, 80 Al of the solution was removed and replaced with 80 Al of fresh PBS. The activity of the collected sample was measured in a scintillation counter. At the final time point, the activity of the remaining solution and the well was read. The percentage of DNA mass released was calculated by dividing the mass released at a given time point by the total mass on the surface. 2.6. Cell culture and transfection The efficiency of gene transfer by the DNA-modified surfaces was examined using the reporter gene luciferase and the cell line HEK293T. PL transfection studies were performed for complexes formed at a charge ratio equal to 5.5:1, and PEI transfection studies were performed using an N/P ratio of 5. HEK293T cells (7000 cells/well) were plated on the modified surfaces and cultured for the indicated times. Soluble PEI (0.4 Al/10 Al TBS) was added to the immobilized DNA/PL complexes for experimental and control conditions to enhance intracellular trafficking [14,15]. No additional soluble PEI was added

to the DNA/PEI immobilized surfaces. Control studies were performed using complexes without biotin groups (DNA/K19, DNA/PEI) that were allowed to adsorb nonspecifically to the substrate. An additional control was performed by adsorption of DNA complexes (DNA/K19, DNA/PEI) to tissue cultures treated at 96-well plates for 2 h. Control substrates were washed with TBS buffer to remove loosely bound complexes. For the PEI/DNA system, an additional control was performed by bolus delivery of DNA complexes (0.16 Ag) to HEK293T cells (5000 cells/ well) 1 day after plating. Cells were cultured at 37 jC and 5% CO2 in DMEM (Invitrogen, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. For measurements of luciferase activity, the cells were lysed and assayed for luciferase enzymatic activity (Promega, Madison, WI) at the indicated times. The luminometer was set for a 3-s delay and an integration of the signal for 10 s. Protein levels of the cell lysate were analyzed (Bradford assay; BioRad, Hercules, CA) at the indicated times. For the time course experiments, complexes were formed with a ratio of K214-B/K19 polymers equal to 5:95 (64 mol biotin/mol DNA). Bolus delivery was performed as a control using DNA/K19 complexes. For the bulk transfection studies, HEK293T cells (5000 cells/well) were plated the day prior to transfection. DNA/K19 complexes were formed at a charge ratio of 5.5:1. The quantity of DNA added as a bolus (0.1 Ag DNA) was equal to the quantity of DNA that bound to the substrate for the 5:95 ratio of K214-B/K19 (i.e., 64 mol biotin/mol DNA). Luciferase expression levels were determined at the indicated time points. 2.7. Statistical analysis The results are representative of replicate experiments performed with a sample size equal to three or four in each experiment, as indicated on the figure legend. The results are represented by the mean and standard deviation. All statistical analyses were performed in the computer program InStat using either the Student’s t test for single comparisons or the Tukey test for multiple comparisons. Probability values less than 0.05 were considered significant.

3. Results 3.1. Polylysine 3.1.1. Biotinylation and DNA complexation PLs (K214, DK214) were covalently modified with biotin residues through the primary amines on the side chains, and retained the ability to complex with DNA. This approach for covalent modification allowed for multiple biotin residues to be attached per PL chain. PLs were biotinylated at ratios of 3.8 [K214-B (3.8)] and 12.9 [K214-B (12.9)] mol biotin/mol PL for K214-B, and 10.9 for DK214-B [DK214-B (10.9)]. The ability of biotinylated PLs to complex with DNA was assessed through the elimination of electrophoretic mobility during gel electrophoresis (Fig. 2). The electrophoretic mobility of DNA was eliminated at a charge ratio ( F ) of 2.4, 3.1, and 3.1 for K214-B (3.8), K214-B (12.9), and DK214-B (10.9), respectively (Fig. 2). Unmodified PLs (K214, DK214) eliminated the electrophoretic mobility at a charge ratio of 1.2 (not shown). Trypsinization of the DNA complexes restored the electrophoretic mobility for the K214-B/DNA complexes but not the DK214-B/ DNA complexes, indicating that K214-B was degraded by the enzyme and released the DNA into the solution.

Fig. 2. Electrophoretic retardation of DNA complexes shows that biotinylated polylysine condenses DNA. Complexes were formed with biotinylated (A) K214-B (3.8), (B) K214-B (12.9), and (C) DK214-B (10.9). The charge ratio ( F 0 – 5.5) is shown on the top of the gels and represents the ratio of lysines/phosphates. The last lane of each gel contains samples that were treated with trypsin (T). Note that for this and all subsequent figures, the charge ratio is calculated based on the nonbiotinylated polymer.

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The number of biotin molecules in a complex can be controlled by mixing biotinylated PL with nonbiotinylated PL. The nonbiotinylated peptide K19 was used because the charge ratio at which the electrophoretic mobility was eliminated (3.1; not shown) was similar to that obtained with the biotinylated peptides, which likely allows incorporation of both peptides into the complex. Mixtures of the biotinylated PL (K214-B, DK214-B) and the low-molecular-weight PL formed complexes with DNA, as evidenced by elimination of electrophoretic mobility (data not shown). Fluorescence microscopy was used to visualize complex binding to the substrate during an initial 2h incubation. A fluorescein isothiocyanate (FITC) hgalactosidase plasmid was complexed with either K214 or K214-B (3.8 mol biotin/mol PL) at a charge ratio ( F ) of 5.5 and incubated on neutravidin substrates (Fig. 3). Note that all references to charge ratio are based on the amine content of unmodified PL, thus the PL content was the same for all studies. For complexation solely with biotinylated polymers, the biotin modification reduces this charge ratio to 5.4 and 5.2 for K214-B (3.8) and K214-B (12.9), respectively. Complexes formed with nonbiotinylated PL (K214/DNA) formed aggregates within 20 min of incubation on the surface (Fig. 3A) and these aggregates increased in size during the incubation. After 120 min (Fig. 3C), the largest aggregates were visible to the naked eye. Conversely, complexes formed with biotinylated PL (K214-B/DNA) were stable throughout the incubation period (Fig. 3D – F). Complexes formed with the biotinylated PL did not aggregate, suggesting that the presence of biotin residues prevented the aggregation of complexes when incubated on a biotin –binding substrate. After the 2-h incubation, the substrates were washed to remove complexes that are not bound to the substrate. The addition of PEI to the tethered complexes, which was done to enhance intracellular trafficking, did not alter the appearance of the complexes on the substrate (not shown). 3.1.2. Charge ratio For NA surfaces, the dependence of substrate binding on the number of biotin groups was subsequently examined for a range of charge ratios. Complexes were formed solely with biotinylated PL [K214-B (3.8) or K214-B (12.9)] at charge ratios

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Fig. 3. Fluorescence photomicrographs of biotinylated and nonbiotinylated DNA/polylysine complexes during incubation on neutravidin surfaces. Nonbiotinylated (A – C) and biotinylated (D – F) K214 was complexed with DNA and incubated on the substrate for 120 min. Images were captured at 20 min (A, D), 40 min (B, E), and 120 min (C, F) of incubation prior to washing the substrate. Images were captured at a magnification of  200.

( F ) of 0.6, 1.8, 3.0, and 5.5. Increasing the charge ratio of DNA/K214-B complexes increased the surface density of DNA. Complexes formed at charge ratios of 0.6 and 1.8 resulted in low surface densities ( < 0.6 Ag DNA/cm2; Fig. 4) for both K214-B tested. Increasing the charge ratio from 1.8 to 3.0 increased complex binding significantly for both biotinylated PLs [ p < 0.05 for K214-B (3.8) and p < 0.001 for K214-B (12.9)]. For complexes formed

with K214-B (12.9), substrate binding was maximal at a charge ratio of 3.0 and a further increase to 5.5 did not increase binding ( p>0.05). For complexes formed with a lower extent of modification [K214-B (3.8)], further increasing the charge ratio from 3 to 5.5 increased substrate binding ( p < 0.001). Subsequent studies were performed at a charge ratio equal to 5.5 since the binding was similar for both polymers [K214-B (12.9) and K214-B (3.8)].

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Fig. 4. Density of substrate-associated DNA/polylysine complexes. Complexes were formed at charge ratios ranging from 0.6 to 5.5 using K214-B (degree of biotinylation equal to 3.8 or 12.9 mol biotin/mol PL). Note that the numbers in parentheses represent the average moles of biotin per mole of DNA. The data are presented as the average F S.D., and the symbols * and *** indicate statistical significance at levels of p < 0.05 and p < 0.001, respectively, for the comparison indicated. p values were obtained using Tukey multiple comparisons tests.

3.1.3. Biotin distribution within the complex The quantity of substrate-associated DNA increased with an increase in the number of biotin residues for complexes formed at a charge ratio equal to 5.5. Nonbiotinylated PLs (K214, K19) used for DNA condensation resulted in low substrate densities ( < 0.25 Ag DNA/cm2) due to nonspecific adsorption when the complexes were incubated on NA surfaces. The presence of biotin residues increased the density of substrate-associated DNA relative to the absence of biotin groups ( p < 0.05). The biotin/DNA molar ratio was manipulated either through the fraction of cationic polymers that were biotinylated (range of 100:0 to 0:100), or the extent of polymer functionalization (3.8 vs. 12.9). Note that the moles of biotin per mole of DNA are indicated in parentheses below each condition in Fig. 5A. Additionally, the ratio of biotinylated PL to nonbiotinylated PL represents the percentage of each based on the total number of lysine residues. Increasing the fraction of biotinylated polymers in the complex led to increasing densities of DNA on the substrate. The maximal density of substrate-associated DNA was observed for complexes formed solely with biotinylated peptides (100:0, p < 0.001) (Fig. 5A). Increasing the number of biotin

residues per mole of PL from 3.8 to 12.9 served to increase the density of DNA on the substrate for all conditions. However, this increase was maximal for complexes formed with a percentage ratio of K214-B/ K19 equal to 50:50 ( p < 0.001). The binding of complexes to tissue culture plastic (TCP), which has been established to nonspecifically adsorb PL, was also examined. K19/DNA complexes incubated in TCP for 2 h resulted in surface densities of 4.7 F 0.79 Ag DNA/cm2 (not shown). Complexes that were tethered to the surface through biotin residues resulted in higher expression levels than nonspecifically adsorbed complexes (Fig. 5B). Transfection by substrate-mediated delivery was quantified by culturing HEK293T (Fig. 5B) cells on substrates with tethered complexes that contained the plasmid encoding for the reporter gene luciferase. With the exception of the substrates with DNA immobilized exclusively with K214-B (3.8), complexes that were specifically tethered to the substrates resulted in significantly higher levels of expression ( p < 0.05) than complexes that were nonspecifically adsorbed to NA substrates or TCP. Interestingly, transfection for nonspecifically adsorbed complexes (no biotin residues) was greater on NA substrates than

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Fig. 5. Density and transfection of substrate-associated DNA/polylysine complexes formed with varying biotin distributions. (A) DNA density and (B) transgene expression for complexes formed at a charge ratio of 5.5:1. Note that the numbers in parentheses represent the average moles of biotin per mole of DNA. The data are presented as the average F S.D., and the symbol * indicates statistical significance at a level of p < 0.05 for the comparisons indicated. p values were obtained using the Student’s t test with single comparisons. The labels NA and TCP indicate the substrate was neutravidin and tissue culture polystyrene, respectively.

on TCP ( p < 0.001), despite the significantly higher density of DNA adsorbed to TCP (4.7 Ag DNA/cm2) than to NA ( < 0.25 Ag DNA/cm2). For complexes specifically tethered to the substrate through biotin residues, increasing the number of biotin residues on the complex had a biphasic effect on transfection, which depended upon their distribution within the complex. The biotin/DNA molar ratio in the complex was manipulated either through the percentage ratio of biotinylated polymers (range of

100:0 to 0:100), or the extent of polymer functionalization (3.8 vs. 12.9 mol biotin/mol K214). Complexes formed with the smallest fraction of biotinylated polymers (5:95) exhibited greater transfection than complexes formed exclusively with biotinylated polymers (100:0, p < 0.001; Fig. 5B). However, increasing the extent of biotinylation for the peptides (3.8 – 12.9) led to increased cellular transfection on the substrate. Complexes tethered with K214-B (12.9) had up to ninefold increase in luciferase expression than com-

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plexes tethered with K214-B (3.8) ( p < 0.05). Increasing the extent of peptide modification can be employed to increase the number of available biotin residues, which may facilitate substrate binding without decreasing cellular transfection. 3.1.4. Transfection with poly-D-lysine (PDL) Cellular transfection by poly-L-lysine (K214) was compared with poly-D-lysine (DK214), a nonenzymatically degradable polymer, to determine if degradation by cellular proteases is the mechanism regulating internalization and transfection. Cellular transfection with DK214 was equal to, or greater than, the transfection using K214 (Fig. 6). The use of DK214 increased transfection by a factor of 5 for complexes formed solely with the biotinylated PLs (100:0) with similar levels of biotinylation ( p < 0.05). For complexes formed with mixtures of biotinylated (K214-B or DK214-B) and nonbiotinylated PLs (K19) at a ratio of 5:95, transfection was not significantly enhanced by using DK214 compared to K214. 3.1.5. Complex release from the substrate The release of complexes from the substrate was performed to examine the stability of complexes on

Fig. 7. Release of substrate-associated DNA/polylysine complexes formed with varying quantities of the biotinylated polymer [K214-B (12.9)]. Release studies were performed with (A) the addition of PEI to the surface (0.4 Al of PEI in 10 Al or TBS) or (B) no addition of PEI to the surface. DNA released was quantified using 32P-labeled DNA. The data are presented as the average F S.D.

Fig. 6. Expression levels by substrate-mediated transfection for complexes formed with enzymatically degradable and nondegradable polylysine. Note that the numbers in parentheses represent the average moles of biotin per mole of DNA. The data are presented as the average F S.D., and the symbol * indicates statistical significance at a level of p < 0.05 for the comparison indicated. p values were obtained using the Student’s t test for single comparisons.

the surface, and the effect of PEI on that stability. These studies also serve to determine if release from the substrate is a possible mechanism affecting transfection. Release studies were performed for complexes with 12.9 biotins per PL, which was the condition that gave the highest levels of transfection. The quantity of DNA released from the surface during the first 24 h ranged from 10% to 20% of the quantity that was immobilized (Fig. 7A and B). For complexes formed at a ratio of 5:95, the addition of PEI to the culture well had no effect on the release rate. For all other conditions, the addition of PEI to the wells

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4 and 8 days for substrate-mediated delivery was significantly greater than expression from bolus delivery at the same time points ( p < 0.01 and p < 0.05, respectively). Note that the quantity of DNA added as a bolus was equal to the quantity of DNA immobilized to the substrate, and that the complexes were formed using either 95% K19 in the case of the tethered complexes or 100% K19 in the case of bolus delivery. 3.2. PEI

Fig. 8. Expression levels by surface-mediated and bulk mediated transfection after 1, 2, 4, and 8 days of transfection. Complexes for substrate-mediated delivery were formed with a biotinylated/ nonbiotinylated polylysine ratio equal to 5:95 (i.e., 64 mol biotin/ mol DNA) and were incubated on NA surfaces. Complexes for bulk-mediated delivery were formed using K19. The quantity of DNA delivered was equivalent to the amount immobilized to the substrate. The data are presented as the average F S.D., and the symbols * and ** indicate statistical significance at levels of p < 0.05 and p < 0.01, respectively, for the given time point. p values were obtained using the Student’s t test for single comparisons.

3.2.1. Complexation and substrate binding The ability of nonbiotinylated and biotinylated linear PEI to condense DNA was assessed through the elimination of electrophoretic mobility using gel electrophoresis. Both nonbiotinylated and biotinylated PEI were able to completely stop the electrophoretic mobility of DNA at an N/P ratio of 1.3 (Fig. 9), indicating that the DNA was fully condensed at the N/ P ratio of 5 used for the binding and transfection studies. Complex formation with biotinylated PEI supported the immobilization to the substrate; however, nonbiotinylated PEI had high levels of nonspecific binding and biotinylation had only modest effects. DNA surface densities were quantified utilizing 32P

decreased the release rate of DNA (Fig. 7A) relative to that obtained without PEI addition (Fig. 7B). Over 30% of the DNA was released over 8 days when PEI was not added, whereas the addition of PEI resulted in approximately 21% of the DNA released. 3.1.6. Expression profile by substrate-mediated delivery Substrate-mediated delivery enhanced the duration of protein production on the substrates relative to the bolus delivery of DNA complexes to the media (Fig. 8). The bolus delivery of DNA complexes to the media, which is the standard transfection procedure, shows increasing luciferase expression during the first 2 days and decreases during the next 6 days. Substrate-mediated delivery shows a similar increase in luciferase expression during the first 2 days; however, luciferase activity at 4 days was similar to that measured at day 2, which then decreases through the 8 days of the study. The transgene expression at

Fig. 9. Electrophoretic retardation of DNA shows that nonbiotinylated PEI and biotinylated PEI condense DNA. Complexes were formed with (A) nonbiotinylated PEI and (B) biotinylated PEI. The charge ratio is shown on the top of the gels and represents the ratio of nitrogen to phosphate.

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complexes (similar to Fig. 3D – F) either in the presence or absence of biotinylated PEI (not shown). 3.2.2. PEI transfection Substrate-mediated transfection by PEI/DNA complexes led to higher levels of transgene expression than that observed with PL/DNA complexes; however, the transfection levels did not vary with the extent of biotinylation. Transfection by substrate-mediated delivery of DNA/PEI complexes was quantified by culturing HEK293T cells on substrates with tethered complexes that contained the plasmid encoding for the reporter gene luciferase. There was no difference in the luciferase expression between the complexes that contained biotin groups and those without, suggesting that transfection was most likely mediated by nonspecifically bound complexes (Fig. 10B). Bolus delivery of PEI/DNA complexes resulted in similar levels of transfection as found with the surface-mediated delivery (Fig. 10B). Adsorption of PEI/DNA complexes to TCP did not mediate transfection, consistent with the observations with PL/DNA (data not shown). 3.2.3. DNA/PEI complex release from the surface Release studies with 32P DNA demonstrated a slow release for all PEI complexes immobilized to the substrate, but the nonbiotinylated complexes had a greater extent of release. Complexes formed with biotinylated PEI had approximately 15% released Fig. 10. Density and transfection of substrate-associated DNA/PEI complexes formed with varying quantities of biotinylated PEI. (A) DNA density and (B) transgene expression for PEI/DNA complexes formed at an N/P ratio equal to 5. Note that the numbers in parentheses represent the average moles of biotin per mole of DNA. The data are presented as the average F S.D., and the symbol **indicates statistical significance at a level of p < 0.01. p value was obtained using Tukey multiple comparisons tests.

DNA. Complexes that were formed solely with biotinylated PEI (100:0) had statistically higher amounts of DNA on the surface than any other condition tested ( p < 0.01; Fig. 10A). Complexes formed with mixtures of biotinylated and nonbiotinylated PEI (50:50 and 5:95) had surface densities equivalent to complexes formed without biotinylated PEI (0:100; Fig. 10A). Additionally, studies performed with fluorescently labeled DNA showed no aggregation of the

Fig. 11. Release of substrate-associated DNA/PEI complexes formed with varying quantities of the biotinylated polymer. DNA released was quantified using 32P-labeled DNA. The data are presented as the average F S.D.

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during the first day and a total of 32% released during the 8-day period (Fig. 11). No differences were observed at any time point for different compositions of biotinylated PEI in the complex. Complexes formed without biotin also had a 15% release after 1 day, yet had a release of 34% and 59% of the immobilized DNA after 4 and 8 days, respectively, which is significantly higher than that observed for the biotinylated complexes ( p < 0.05).

4. Discussion The immobilization of DNA complexes to a substrate that supports cell adhesion has the potential to enhance gene transfer by maintaining DNA within the cellular environment while limiting clearance and complex aggregation. The objective of this report has been to examine the design of the tethering system (e.g., extent of functionalization) and the properties of the cationic polymer for its impact on complex binding to the substrate and the subsequent cellular transfection. DNA is complexed with cationic polymers (PL, PEI), a fraction of which is functionalized with biotin, which permits binding to a NA substrate. Surfaces densities ranging from 0.4 to 2.6 Ag DNA/ cm2 were obtained for the PL system and from 0.7 to 1.0 Ag DNA/cm2 for the PEI system, with greatest surface densities observed for complexes with the largest number of biotin residues. The number of biotin groups for the PL system had a dual effect on cellular transfection, which depended upon their distribution in the complex. Increasing the fraction of PL with biotin residues decreased luciferase activity; however, increasing the number of biotin residues on each PL led to increases in luciferase activity. For PEI transfection from the substrates, the number of biotin groups present on the complex did not affect transgene expression. Release studies demonstrated that approximately 20 –30% of the immobilized DNA was released from the substrate during the 8 days, with 10 – 20% released during the first 24 h. The transfection studies using enzymatically degradable (PL) and nondegradable (PDL, PEI) polymers suggest that degradation of the polymer for the release of complexes for internalization is not required for transfection. Additionally, the duration of transgene expression was extended for surface-mediated trans-

fection when compared to bulk-mediated transfection for the PL system. The binding of DNA/cationic polymer complexes to the substrate results from a combination of specific and nonspecific interactions. In this system, we define specific binding as the interaction between the biotin residues attached to the cationic polymers with the NA-containing substrate, which had been treated to reduce nonspecific binding. PL (primary amines) and linear PEI (primarily secondary amines) that were modified with biotin residues were investigated for their specific and nonspecific binding to the substrate. Complexes formed with nonbiotinylated PL had significantly less nonspecific adsorption of complexes than complexes formed with nonbiotinylated PEI (Figs. 4 and 9A). Increased protein binding has been observed for surface-modified nanospheres containing secondary amines relative to those containing primary amines [16], which may have increased the nonspecific binding of the PEI/DNA complexes. Interestingly, substrate immobilization of PL/DNA and PEI/ DNA complexes inhibits their aggregation, which can occur rapidly both in vivo and in vitro and reduces the activity of DNA complexes [17]. PL/DNA complexes aggregate in solutions with salt concentrations of 150 mM [18,19] and/or serum proteins such as albumin [20,22]. Linear PEI complexed with DNA is observed to increase in size during the initial hours of complex formation [23]. Several strategies to limit complex aggregation in polyelectrolyte solutions are being developed [17] such as steric stabilization [21 – 27], caging [19,28], and anionic polymer/DNA particles [29,30]. Immobilization of DNA complexes to a substrate represents another potential strategy to prevent aggregation of the DNA complexes. For specific binding of biotinylated PL/DNA complexes, the amount of immobilized complexes and the efficiency of transgene expression are dependent on the distribution of biotin within the complex. The incorporation of biotin groups into the complex was increased through either increasing the percentage of PL chains with biotins, or increasing the extent of biotinylation per polymer. Importantly, the binding affinity of these different cationic polymers for the DNA must be balanced to ensure that the biotin groups are incorporated into the complex; otherwise, substrate binding is low [13]. The affinity of K214-B for DNA is equal to, or greater than, that of K19 as

determined by the ability of PL to eliminate the mobility of DNA during gel electrophoresis. Although increasing numbers of biotin residues enhanced substrate binding (Fig. 5A), the distribution of these residues within the complex was an important determinant of transfection (Fig. 5B). Transfection was enhanced only when the extent of polymer biotinylation was increased from 3.8 to 12.9 (Fig. 5B), and not when the percentage of biotinylated polymers was increased. The increased transfection likely results from the combination of an increased density of DNA that was immobilized to the substrate and a decreased number of cationic polymers in the complex that are interacting with the substrate. Cationic polymers modified with 12.9 biotin residues were able to achieve maximal binding of complexes at a charge ratio of 3.0, whereas polymers modified to a lesser extent (3.8) achieved maximal surface density at a higher charge ratio (5.5) (Fig. 4). Thus, fewer numbers of cationic polymers are required to achieve maximal binding for the larger extent of modification. A smaller fraction of cationic polymers interacting with the substrate enhances transfection (compare K214-B with K214-B/K19; Fig. 5B), which may result from an increased rate of release from the substrate and/or internalization of the complex by the cell. Taken together, the binding and transfection results suggest that the extent of functionalization of the cationic polymer and the fraction of modified polymers in the complex must be balanced to maximize substrate binding while not restricting cellular internalization. Transfection levels using PEI as the condensing polymer resulted in approximately twofold higher levels of transgene expression than using PL as the condensing polymer (Figs. 4 and 9B). The immobilization of PEI/DNA complexes produced transfection that was independent of the biotin content, suggesting that the nonspecific interaction between the secondary amines and the NA surface regulated the transfection. For PL complexes immobilized to the substrate, PEI was added to the media to facilitate transfection. PEI and PEI/DNA complexes have been added to PL complexes to facilitate transgene expression, likely by enhancing intracellular trafficking [15]. Many investigators have used chloroquine to facilitate the intracellular trafficking of PL/DNA; however, prolonged exposure to chloroquine (>4 h) can be cyto-

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toxic. Internalization of complexes from the substrate may occur throughout the culture period, thus a short incubation period with chloroquine was not expected to be effective for this system. Soluble addition of PEI did not affect the PL/DNA size and actually decreased the rate of complex release from the substrate (Fig. 6). Thus, specific binding may be achieved through the use of PL/DNA complexes, and transfection can be enhanced by the addition of a secondary polymer to facilitate intracellular trafficking events. Alternatively, branched PEI, which contains primary, secondary, and tertiary amines in a 1:2:1 ratio, may provide a means to obtain specific binding and to maintain transfection levels. Internalization of DNA complexes from the substrate must occur either by release of the complexes from the substrate, or by direct internalization of the immobilized complexes. The release rate of complexes from the surface was studied using radiolabeled DNA (32P DNA). Release from the surface may be mediated by enzymatic degradation of PL or neutravidin, desorption of NA from the surface, or dissociation of the biotin – NA interaction. The PL and NA proteins may be digested by cell-releasing proteases, causing the release of the complexes. The similar levels of transfection obtained with PL and PDL suggest that degradation of the cationic polymer is not a limiting step (Fig. 6). NA desorption from the surface is possible; however, the method by which the surfaces were prepared was not disclosed by the manufacturer. Biotin –avidin is a strong noncovalent interaction and the large number of interactions between the complex and the substrate is unlikely to result in the release of the complex. For complexes formed with K214-B (12.9), approximately 24% and 31% of the complexes were released for biotinylated/ nonbiotinylated ratios polymer equal to 100:0 and 5:95, respectively (Fig. 7). These percentages correlate to 156 ng (100:0) and 53 ng (5:95) of DNA. Transgene expression, however, was significantly higher for the 5:95 ratio, which had lower amounts of DNA released. For DNA complexed with PEI, approximately 33% and 59% of the DNA were released from complexes formed at biotinylated/nonbiotinylated ratios equal to 100:0 and 0:100, which corresponds to 57 and 80 ng DNA for the two cases. Transfection by released complexes may be mediating the transfection; however, the extent of transfection

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does not directly correlate with the quantity of DNA released. The alternative hypothesis to release from the substrate involves internalization of the DNA directly from the substrate, which could occur by the process of vector unpacking. Vector unpacking represents the dissociation of the DNA from the cationic polymer and is an important step that must occur intracellularly to facilitate expression of the DNA [31]. A partial unpacking process may occur extracellularly, in which DNA complexes dissociate from the substrate-bound cationic polymer to enable cellular internalization. Between 70% and 80% of the immobilized biotinylated complexes remains associated with the substrate through 8 days of the release study. Development of the system to allow cellular internalization directly from the substrate, rather than following release, may provide a means to regulate delivery. Substrate-mediated delivery provides equivalent or greater levels of transfection relative to the bolus delivery of similar quantities of DNA complexes. Previous studies using the reporter gene b-galactosidase demonstrated that substrate-mediated delivery results in transfection only in the location at which DNA had been immobilized [13]; however, delivery of the DNA complexes as a bolus results in cellular transfection throughout the cell population. In this report, transfection studies employed the reporter gene luciferase, which can characterize changes in promoter activity. The substrate-associated complexes had a composition similar to the complexes delivered as a bolus [differing only by 5% K214-B (3.8) for substrateimmobilized complexes]. Bolus delivery resulted in maximal expression at 2 days, with expression decreasing by an order of magnitude throughout the remainder of the study (Fig. 6). However, maximal expression levels for substrate-mediated delivery of equivalent DNA quantities were observed through days 2 – 4 of culture, and the subsequent decrease through the remainder of the study was less than that observed by bolus delivery. Different temporal patterns of gene expression have been observed for complexes formed with a mixture of functionalized and nonfunctionalized complexes, which may result from differences in complex stability or vector unpacking [32]. For substrate-tethered complexes, the possibility also exists that the extended expression of luciferase results from different patterns of internali-

zation. The enhanced gene expression may result from the activity of the complexes being maintained for extended times since the substrate-tethered complexes cannot aggregate. Importantly, DNA may be internalized from the substrate at later times of culture, and not only at the initial exposure. The transfection studies on NA substrates and tissue culture polystyrenes (TCPs) also suggest that the interaction between the substrate and the DNA complex must be balanced to support both immobilization and internalization. Tissue culture plastic is commonly coated with proteins, such as PL, to support cell attachment. Complexes incubated on TCP resulted in a density of 4.7 F 0.8 Ag DNA/cm2, which likely resulted from the nonspecific interactions between the PL and the culture substrate. Nevertheless, transfection on these substrates was low (Fig. 4). Similarly, DNA/PEI complexes nonspecifically adsorbed to TCP also resulted in very low levels of expression. The high density of immobilized DNA suggests that there are extensive interactions between the cationic polymer and the substrate, which may limit cellular internalization, or disrupt the structure of the complexes. The NA surfaces, which are treated to reduce nonspecific adsorption, did allow some limited nonspecific adsorption of nonbiotinylated PL complexes ( < 0.25 Ag DNA/cm2). Despite the significantly lower densities of DNA on these surfaces, the NA substrates exhibited significantly higher levels of transfection than the TCP from nonspecifically adsorbed complexes. The specific binding of the biotinylated PL complexes to the NA surfaces increased transfection up to 40-fold for levels of substrate-associated DNA similar to that observed for nonbiotinylated complexes. For PEI complexes, nonspecific interactions (i.e., not biotin – NA) between the linear PEI seemed to dominate the immobilization and produced similar levels of transfection for all cases.

5. Conclusion Substrate-mediated delivery of nonviral DNA complexes may have implications for both basic sciences and clinical applications. This method of gene delivery represents a synthetic approach for nonviral complexes that mimic the binding of retroviruses to fibronectin, which can enhance viral gene transfer [9,10]. Recently,

this concept of matrix immobilization has been extended to the adenovirus system by immobilizing antibodies to a collagen gel that binds the virus [11]. The immobilized adenovirus resulted in site-specific localization of transgene expression and can avoid distal side effects. This synthetic approach for the immobilization of nonviral complexes may provide a more versatile approach to regulate the attachment of DNA to a substrate and subsequent cellular internalization. These studies demonstrate that the cationic polymer, the substrate, and the tethering scheme can impact substrate binding, cellular internalization, and, ultimately, transgene expression. Appropriate design of these features may permit transfection to be controlled at the material surface. In wound healing and tissue regeneration, immobilization of DNA complexes to a biocompatible, biodegradable matrix could be used to augment the natural cellular processes. Alternatively, substrate-mediated delivery could be applicable to basic science research, such as the creation of transfected cell arrays [12,33]. Acknowledgements This work was supported, in part, by grants from the NSF (L.D.S.), the Specialized Program of Research Excellence (SPORE) in Breast Cancer (grant no. P50CA89018) of the National Cancer Institute (L.D.S.), and the Northwestern Biotechnology Training grant (T.S.). L.D.S. is a member of the Robert H. Lurie Comprehensive Cancer Center. We would like to thank Peter Chung, Zain Bengali, and Brian C. Anderson (Northwestern University) for technical assistance. References [1] A.V. Kabanov, V.A. Kabanov, DNA complexes with polycations for the delivery of genetic material into cells, Bioconjug. Chem. 6 (1) (1995) 7 – 20. [2] F.D. Ledley, Pharmaceutical approach to somatic gene therapy, Pharm. Res. 13 (11) (1996) 1595 – 1614. [3] W.C. Tseng, F.R. Haselton, et al., Transfection by cationic liposomes using simultaneous single cell measurements of plasmid delivery and transgene expression, J. Biol. Chem. 272 (41) (1997) 25641 – 25647. [4] L.D. Shea, E. Smiley, et al., DNA delivery from polymer matrices for tissue engineering, Nat. Biotechnol. 17 (6) (1999) 551 – 554.

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[5] D. Luo, W.M. Saltzman, Enhancement of transfection by physical concentration of DNA at the cell surface, Nat. Biotechnol. 18 (8) (2000) 893 – 895. [6] W.C. Tseng, F.R. Haselton, et al., Mitosis enhances transgene expression of plasmid delivered by cationic liposomes, Biochim. Biophys. Acta 1445 (1) (1999) 53 – 64. [7] J. Fang, Y.Y. Zhu, et al., Stimulation of new bone formation by direct transfer of osteogenic plasmid genes, Proc. Natl. Acad. Sci. U. S. A. 93 (12) (1996) 5753 – 5758. [8] J. Bonadio, E. Smiley, et al., Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration, Nat. Med. 5 (7) (1999) 753 – 759. [9] D.A. Williams, Retroviral – fibronectin interactions in transduction of mammalian cells, Hematop. Stem Cells (1999) 109 – 114. [10] I. Julkunen, T. Vartio, et al., Localization of viral-envelopeglycoprotein-binding sites in fibronectin, Biochem. J. 219 (2) (1984) 425 – 428. [11] R.J. Levy, C. Song, et al., Localized adenovirus gene delivery using antiviral IgG complexation, Gene Ther. 8 (9) (2001) 659 – 667. [12] K. Honma, T. Ochiya, et al., Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions, Biochem. Biophys. Res. Commun. 289 (5) (2001) 1075 – 1081. [13] T. Segura, L.D. Shea, Surface-tethered DNA complexes for enhanced gene delivery, Bioconjug. Chem. 13 (3) (2002) 621 – 629. [14] R.P. Harbottle, R.G. Cooper, et al., An RGD-oligolysine peptide: a prototype construct for integrin-mediated gene delivery, Hum. Gene Ther. 9 (7) (1998) 1037 – 1047. [15] A. Kichler, C. Leborgne, et al., Polyethylenimine-mediated gene delivery: a mechanistic study, J. Gene Med. 3 (2) (2001) 135 – 144. [16] A. Gessner, A. Lieske, et al., Functional groups on polystyrene model nanoparticles: influence on protein adsorption, J. Biomed. Mater. Res. 65A (3) (2003) 319 – 326. [17] J.E. Hagstrom, Self-assembling complexes for in vivo gene delivery, Curr. Opin. Mol. Ther. 2 (2) (2000) 143 – 149. [18] C. Plank, M.X. Tang, et al., Branched cationic peptides for gene delivery: role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes, Hum. Gene Ther. 10 (2) (1999) 319 – 332. [19] V.S. Trubetskoy, A. Loomis, et al., Caged DNA does not aggregate in high ionic strength solutions, Bioconjug. Chem. 10 (4) (1999) 624 – 628. [20] P. Lucas, D.A. Milroy, et al., Pharmaceutical and biological properties of poly(amino acid)/DNA polyplexes, J. Drug Target. 7 (2) (1999) 143 – 156. [21] D. Oupicky, K.A. Howard, et al., Steric stabilization of poly-Llysine/DNA complexes by the covalent attachment of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide], Bioconjug. Chem. 11 (4) (2000) 492 – 501. [22] B. Xu, S. Wiehle, et al., The contribution of poly-L-lysine, epidermal growth factor and streptavidin to EGF/PLL/DNA polyplex formation, Gene Ther. 5 (9) (1998) 1235 – 1243. [23] D. Goula, J.S. Remy, et al., Size, diffusibility and transfection performance of linear PEI/DNA complexes in the

GENE DELIVERY

T. Segura et al. / Journal of Controlled Release 93 (2003) 69–84

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84

[24]

[25]

[26]

[27]

[28]

T. Segura et al. / Journal of Controlled Release 93 (2003) 69–84 mouse central nervous system, Gene Ther. 5 (5) (1998) 712 – 717. M. Ogris, S. Brunner, et al., PEGylated DNA/transferrin – PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery, Gene Ther. 6 (4) (1999) 595 – 605. M. Ogris, P. Steinlein, et al., DNA/polyethylenimine transfection particles: influence of ligands, polymer size, and PEGylation on internalization and gene expression, AAPS PharmSci. 3 (3) (2001) E21. M.A. Wolfert, P.R. Dash, et al., Polyelectrolyte vectors for gene delivery: influence of cationic polymer on biophysical properties of complexes formed with DNA, Bioconjug. Chem. 10 (6) (1999) 993 – 1004. V. Toncheva, M.A. Wolfert, et al., Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers, Biochim. Biophys. Acta 1380 (3) (1998) 354 – 368. R.C. Adami, K.G. Rice, Metabolic stability of glutaraldehyde

[29]

[30]

[31]

[32]

[33]

cross-linked peptide DNA condensates, J. Pharm. Sci. 88 (8) (1999) 739 – 746. V.S. Trubetskoy, V.G. Budker, et al., Self-assembly of DNA – polymer complexes using template polymerization, Nucleic Acids Res. 26 (18) (1998) 4178 – 4185. V.S. Trubetskoy, A. Loomis, et al., Layer-by-layer deposition of oppositely charged polyelectrolytes on the surface of condensed DNA particles, Nucleic Acids Res. 27 (15) (1999) 3090 – 3095. D.V. Schaffer, N.A. Fidelman, et al., Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery, Biotechnol. Bioeng. 67 (5) (2000) 598 – 606. A.G. Ziady, T. Ferkol, et al., Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo, J. Biol. Chem. 274 (8) (1999) 4908 – 4916. J. Ziauddin, D.M. Sabatini, Microarrays of cells expressing defined cDNAs, Nature 411 (6833) (2001) 107 – 110.