Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons

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Biomaterials 28 (2007) 1443–1451 www.elsevier.com/locate/biomaterials

Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons Jieming Zenga, Xu Wanga, Shu Wanga,b, a

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669 b Department of Biological Sciences, National University of Singapore, Singapore Received 3 August 2006; accepted 15 November 2006 Available online 6 December 2006

Abstract Chemical conjugation of targeting ligands to polycation/plasmid DNA complexes has been widely used to improve the transfection efficiency of nonviral gene delivery vectors. However, conjugation reactions may reduce or even inactivate the biological activities of chemically sensitive moieties, such as proteins and peptides. Here we describe a new method for introducing targeting ligands into nonviral vectors, in which ternary complexes are formed via charge interactions among polyethylenimine (PEI) of 600 Da, plasmid DNA and targeting peptides with positively charged DNA-binding sequence. Owing to the nerve growth factor (NGF) loop 4 hairpin motif in the targeting peptide, these ternary complexes are capable of mediating gene delivery efficiently and specifically into cells expressing the NGF receptor TrkA. In in vitro experiments, the complexes improved luciferase reporter gene expression by up to 1000-fold while comparing with that produced by complexes with nontargeting control peptide. In an in vivo experiment, the ternary complexes with the targeting peptide was 59-fold more efficient than the control ternary complexes in transfecting dorsal root ganglia (DRG), the peripheral nervous sites with TrkA-expressing neurons. In a cell viability study, the ternary complexes were remarkably different from DNA complexes by PEI of 25 kDa, the gold standard for nonviral gene carriers, displaying no toxicity in tested neuronal cells. Thus, this study demonstrates an alternative method to construct nonviral delivery system for targeted gene transfer into neurons. r 2006 Elsevier Ltd. All rights reserved. Keywords: Nonviral vectors; Targeted gene delivery; Neurons; TrkA receptor

1. Introduction For human gene therapy, gene delivery vectors are modified by integrating ligands that recognize particular types of cellular receptors to achieve cell-specific targeting [1–4]. Such a delivery strategy also improves the efficiency of gene transfer by enhancing the entry of gene vectors into the desired cells and reducing the uptake by nontarget cells. Typically, targeting moieties of natural or synthetic origin are chemically conjugated to nonviral DNA carriers. For example, a variety of targeting ligands have been attached Corresponding author. Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Singapore 138669. Tel.: +65 6824 7105; fax: +65 6478 9083. E-mail address: [email protected] (S. Wang).

0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.11.015

covalently to cationic polymer polyethylenimine (PEI) to produce targeted nonviral vectors, including galactosylated PEI to target hepatocytes [5–7], RGD-containing peptide conjugated onto PEI to target integrins on cell surface [8], mannosylated PEI to target dendritic cells [9], transferrin conjugated to PEI to target transferrin receptors [10,11], and EGF conjugated to PEI for enhanced uptake into epithelial cells [12]. The attachment of the targeting moieties to PEI has been achieved via the formation of either stable covalent bonds, disulfide bonds or covalent bonds susceptible to enzymatic cleavage [13–17]. These processes require chemical reactions that may inactivate the biological functions of chemical-sensitive targeting ligands. In one of our previous studies [18], we produced a recombinant polypeptide comprising a histone H1 DNAbinding domain and a cell-targeting domain derived from

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the loops 1 and 2 of nerve growth factor (NGF) in Escherichia coli and demonstrated that it directed plasmid DNA to NGF receptor TrkA-positive cells. We further designed and chemically synthesized a short peptide for targeted gene delivery into TrkA-positive cells [19], in order to construct a delivery vector comprised purely of synthetic materials without the use of living cells or organisms. This peptide, designated as NL4-10K, is composed of a targeting moiety derived from the NGF loop 4 hairpin motif (NL4) and a DNA-binding moiety of 10 lysine residues (10K). We demonstrated that NL4-10K/DNA complexes mediated gene transfer specifically in the cultured cells expressing TrkA receptors [19]. However, transfection was strongly dependent on the addition into the culture medium of chloroquine, a weak base that inhibits fusion of the endosome with the lysosome, thus facilitating endosomal escape of plasmid DNA [19]. While effective in vitro, the chloroquine co-treatment is not a feasible approach for in vivo gene delivery. We reason that protonable amines in PEI polymers may be used to replace chloroquine. Furthermore, including PEI in NL4-10K/ DNA complexes can reduce the amount of the peptide needed for DNA condensation, thus reducing possible side effects of the peptides, such as triggering immune response during in vivo application. In the current study, we wished to determine whether noncovalent bonding of cell targeting moieties to polymer/ DNA complexes through charge interaction can be used as an alternative to covalent chemical conjugation to generate targeted gene delivery systems. Our approach was to make use of the positively charged amino acid residues in NL4-10K peptide for electrostatic interaction with negatively charged plasmid DNA that was partially packed with PEI first. In addition, we wished to study whether such ternary complexes can be used for in vivo gene transfer. We demonstrated that the targeting effect of the NL4-10K peptide was well preserved in self-assembled ternary complexes. 2. Materials and methods 2.1. Peptides, polymers and plasmid DNA NL4-10K is a 39-amino acid peptide containing a NGF loop 4 hairpin motif of 29 amino acids followed by a 10-lysine sequence. The peptides were used as TrkA-targeting peptides and described in our previous work [19]. In brief, NL4-10K was produced using a conventional solid-phase, chemical synthetic method and cyclized with a disulfide bond formed between two cysteines in the sequence (Cambridge Research Biochemicals, Cleveland, UK). A 10-lysine peptide, designated as 10K, was obtained from Bio-Synthesis (Lewisville, TX, USA) and used as a peptide control without the targeting moiety. Branched PEI polymers with molecular weight (MW) of 600 Da (PEI600) and 25 kDa (PEI25k) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The stock solutions of PEI600 and PEI25k were prepared in 5% glucose at a concentration that contains 0.1 M of amine nitrogen (‘‘N’’ moiety) [20]. pCAGluc (kindly provided by Yoshiharu Matsuura, National Institute of Infectious Diseases, Tokyo, Japan) was used in in vitro experiments. This reporter plasmid contains a gene encoding firefly luciferase driven by a composite CAG promoter. pCMV C/P-luc constructed previously in our

lab [21] was used in in vivo experiments. This plasmid uses a neuronspecific promoter, human PDGF-b promoter, augmented by the CMV enhancer to drive the expression of firefly luciferase gene. Plasmid DNA was amplified in E. coli and purified with HiSpeed Plasmid Kit (Qiagen, Hilden, Germany). The quantity and quality of the purified plasmid DNA were assessed by optical density at 260 and 280 nm and by electrophoresis in 1% agarose gel. The purified plasmid DNA was dissolved in TE buffer and kept in aliquots at a concentration of 1 mg/ml.

2.2. Preparation and characterization of ternary complexes Polymer/DNA/peptide ternary complexes were prepared at various nitrogen/phosphate ratios, which was defined as N/P ratio and used as a measurement of charge balance in ternary complexes. For the purpose of calculating an N/P ratio, the number of basic amino acid residues in the 10-lysine domain of NL4-10K peptides was taken as the number of ‘‘N’’ moieties per peptide molecule. The required amount of DNA was calculated by taking into account that 1 mg DNA contains 3 nmol of phosphate. To form ternary complexes, the stock solution of PEI600, plasmid DNA and peptides were diluted in a 5% glucose solution to appropriate concentration of each component. The PEI600 solution was then added to the DNA solution at the indicated ratios while vortexing. After 30 min incubation, the solution containing peptides under testing was added to the complexes at indicated ratios to form ternary complexes. The complexes were incubated for another 30 min before being used for experiments. Similarly, PEI25k/DNA complexes at indicated N/P ratios were prepared and used as a positive control in some assays. For agarose gel electrophoresis, polymer/DNA/peptide complexes mixed with a loading buffer were loaded onto an ethidium bromidecontaining 0.7% agarose gel. Gel electrophoresis was run at room temperature in TEB buffer at 80 V for 50 min. DNA bands were visualized by a UV (254 nm) illuminator. To measure the zeta potential and particle size of complexes, complexes were diluted to 1.5 ml with 1 mM KCl buffer to ensure that the measurements were made under conditions of low ionic strength where the surface charge can be accurately measured. The zeta potentials of the complexes were then analyzed by phase analysis light scattering using a Brookhaven ZetaPALS zeta potential analyzer (Brookhaven Instruments Corporation, USA). Default settings on the ZetaPALS were used, i.e. dielectric constant, refractive index and viscosity were assumed to be the same as for water, and the Smoluchowski approximation was used. Determinations were carried out at 22 1C and all buffer solutions were filtered through a 0.22 mM filter before use. For size measurement, the complexes were prepared in the same manner as for zeta potential measurement and then diluted to 3 ml with 1 mM KCl buffer. Determinations were carried out at 22 1C at a fixed angle of 901 by dynamic light scattering using a Brookhaven ZetaPALS submicron particle size analyzer (Brookhaven Instruments Corporation).

2.3. Cell culture Two TrkA-expressing NIH3T3 cell lines, E25 and TRK1, and parental NIH3T3 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich). E25 was developed by Stuart Decker [22], and TRK1 developed by Cordon-Cardo et al. [23], both of which were kindly provided by Dr. Alonzo H. Ross (University of Massachusetts Medical School). To maintain the expression of TrkA in NIH3T3.E25 and NIH3T3.TRK1, 0.5 mg/ml G418 (Sigma-Aldrich) or 50 U/ml hygromycin B (Calbiochem, La Jolla, CA, USA) was used, respectively. A PC12 cell line was obtained from ATCC (Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% FBS and 5% horse serum (Sigma-Aldrich). All the above-mentioned cells were maintained in a humidified incubator with 5% CO2 at 37 1C. Primary cultures of cortical neurons were established from the cortex of embryonic Wistar rats at gestational day 20. Meninges-free cortices were dissected and individual cells were dispersed mechanically by trituration of minced tissue in 3 ml DMEM supplemented with 2% FBS. The suspension

ARTICLE IN PRESS J. Zeng et al. / Biomaterials 28 (2007) 1443–1451 was allowed to settle in a centrifuge tube. The cells in the supernatant were collected by centrifugation at 1000 rpm for 5 min and resuspended gently in DMEM with 10% FBS. Viability of the cells was assessed prior to plating using trypan blue. To obtain neurons, cells were plated onto microplate pre-coated with poly-L-lysine/laminin at a density of 7.5  105 viable cells/cm2. After 2-h incubation to allow neurons to attach, the medium and unattached cells were removed and serum-free DMEM/ F12 medium with 1% N2 supplement (Invitrogen, Carlsbad, CA, USA) was added into the plate. The neurons were then incubated at 37 1C in 5% CO2 in a humidified incubator for 2–5 days before use for experiments.

2.4. Gene transfection For in vitro transfection, the cells were split one day prior to transfection and plated in 48-well plates at a density of 2.5  104 cells/ well. Before transfection, the cell culture medium was replaced with OPTIMEM (Invitrogen). The cells were transfected with gene delivery complexes containing 0.5 mg of plasmid DNA for 4 h. The transfection medium was then replaced with normal culture medium and the treated cells were cultured for another 24 h. The cells were then washed and lysed with the reporter lysis buffer (Promega, WI, USA). The luciferase activity in cell extracts was measured using a luciferase assay kit (Promega). Each measurement was carried out for 10 s in a single-well luminometer (Berthold Lumat LB 9507, Germany). The relative light units (RLU) were normalized by the total protein concentration of the cell extracts, measured with a Dc protein assay kit (Bio-Rad, Hercules, CA, USA). For in vivo experiments, adult male Wistar rats, weighting 250–300 g, supplied by the Laboratory Animal Center, National University of Singapore, were used throughout the study. Before each intrathecal injection, the animals were anesthetized by an i.p. injection of sodium pentobarbital (60 mg/kg of body weight). The back skin of the rats was incised and the spinal column was exposed. The intraspinal space between lumbar vertebrae 3 and 4 (L3 and L4) was chosen as the site of injection with a 100 ml micro-syringe connected with a 26-gauge needle. Slight movements of the tail indicated proper insertion of the needle into the subarachnoid space. For each rat, 50 ml of 5% glucose solution containing 3 mg of plasmid DNA (pCMV E/P–luc) [21] complexed with PEI600 and peptides were injected slowly over 5 min. After the injection, the needle remained in situ for 2 min before being withdrawn. The skin was closed with surgical clips. In the handling and care of animals, the Guidelines on the Care and Use of Animals for Scientific Purposes issued by National Advisory Committee for Laboratory Animal Research, Singapore was followed. The experimental protocol was approved by Institutional Animal Care and Use Committee (IACUC), National University of Singapore and Biological Resource Center, the Agency for Science, Technology and Research (A* STAR), Singapore.

2.5. Cell viability assay For cell viability assay, the cells (10,000 cells/well) were seeded into 96well microtiter plates. The cells were then incubated in culture media containing gene delivery complexes formed at various charge ratios for 48 h. About 20 ml of sterile filtered MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5diphenyl tetrazolium bromide) (5 mg/ml) stock solution in PBS was added to each well reaching a final concentration of 0.5 mg/ml. After 4 h, unreacted dye was removed by aspiration. The formazan crystals were dissolved in 100 ml DMSO per well and measured spectrophotometrically in an ELISA plate reader (Model 550, Bio-Rad) at a wavelength of 595 nm. The spectrophotometer was calibrated to 0 absorbance using culture medium without cells. The cell survival in the presence of complexes was expressed as percentage of cell survival in absence of complexes.

2.6. Immunohistochemistry For immunohistochemistry, rats under anesthesia were perfused with 0.1 M PBS followed by 2% paraformaldehyde in 0.1 M PBS. Lumbar spinal

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cords and DRG at L3–L5 were removed and post-fixed in the same fixative for 2–4 h. Then the samples were transferred into 0.1 M PBS containing 15% sucrose and kept overnight at 4 1C. Frozen sections were cut at 30 mm thickness and mounted on slides. Sections were washed for 20 min in 0.1 M PBS and then incubated overnight with anti-OX42 monoclonal antibody (Harlan Sera Lab, Leicestershire, UK; dilution 1:500). After incubation, sections were rinsed in PBS for 15 min and reacted with Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) against mouse IgG for 1 h. They were then treated with 3,30 diaminobenzidine tetrahydrochloride (Sigma-Aldrich) as a peroxidase substrate. The sections were counterstained with 1% methyl green, dehydrated and mounted in Permount. Similarly, frozen sections of DRGs were prepared and incubated overnight with a polyclonal antibody against luciferase (Promega; dilution 1:150) or a monoclonal antibody against NeuN (Chemicon International, USA; dilution 1:500), followed by the secondary antibody incubation using anti-rabbit IgG TRITC conjugate (Sigma-Aldrich; dilution 1:100) or anti-mouse IgG FITC conjugate (Sigma-Aldrich; dilution 1:100) for 1 h. The processed sections were examined with an Olympus 500 confocal laser scanning microscope.

3. Results 3.1. Formation of PEI600/DNA/NL4-10K ternary complexes As a starting point, we tested different ratios in the preparation of PEI600/DNA complexes in order to generate those with a weak positive surface charge that could be used as a platform for the addition of targeting moiety NL4-10K. Table 1 shows that at an N/P ratio of 5, the surface charge of PEI600/DNA complexes was 6 mV, close to neutrality. PEI600/DNA complexes with an N/P ratio higher than 5 had the surface charge ranged from 21 to 27 mV, which could be high enough to prevent the effective binding of NL4-10K to the complexes. At an N/P ratio lower than 5, there were no enough PEI600 polymers to condense plasmid DNA, thus the size distribution of complexes broadened considerably and we failed to obtain a stable value of particle size. As such, the N/P ratio of 5 between PEI600 and plasmid DNA was chosen for further studies. We then investigated whether NL4-10K was able to bind to the above PEI600/DNA complexes to form noncovalent ternary complexes. We mixed plasmid DNA with PEI600 first, incubated for 30 min and then added various amounts of the peptide at N/P ratios between peptide and DNA from 0 to 5 (the ‘polymer first’ method). In DNA retardation assay (Fig. 1A), although significant reduction of DNA mobility occurred when PEI600 alone was used at an N/P ratio of 5, there were still some plasmid DNA molecules that were not compacted. The negative charges of these DNA molecules were completely neutralized when NL4-10K was added at an N/P ratio of 3 or above. In consistence with the results from the DNA retardation assay, the surface charge of the PEI600/DNA complexes increased from 6 to 22 mV after NL4-10K was added at an N/P ratio of 2.5 and there was no further significant increase with addition of more peptides (Table 1). The complex size decreased from 445 nm for the PEI600/DNA complexes to 180 nm after addition of NL4-10K at an N/P

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Table 1 Zeta potential and particle size measurementa Complexes

N/P ratio

Zeta potential (mV)

Particle size (nm)

PEI600/DNA PEI600/DNA PEI600/DNA PEI600/DNA PEI600/DNA PEI600/DNA/NL4-10K PEI600/DNA/NL4-10K PEI600/DNA/NL4-10K NL4-10K/DNA/PEI600 PEI600/DNA/10K PEI25k/DNA PEI25k/DNA

1/1 5/1 10/1 20/1 30/1 5/1/2.5 5/1/5 5/1/10 5/1/5 5/1/5 10/1 20/1

17.370.5 6.270.7 21.370.9 27.171.5 27.473.1 22.071.0 23.271.1 25.372.1 21.771.8 23.470.8 42.173.3 43.872.8

—b 445.07166.4 122.171.4 141.773.7 135.274.7 193.475.7 180.475.5 168.875.9 176.371.4 175.075.1 191.579.7 194.278.1

a Zeta potential (mV) and particle size (nm) in 1 mM KCl. To prepare most types of ternary complexes, plasmid DNA (20 mg) was packed with PEI polymers first, following by addition of peptides at the indicated N/P ratios. To prepare NL4-10K/DNA/PEI600 complexes, DNA was packed with the peptide first, followed by addition of PEI600. b Probably due to being unable to form stable complexes, considerable discrepancy in size distribution was observed.

ratio of 5, indicating DNA binding and condensation effects of the peptides. These results confirm that peptides with a DNA binding moiety could bind to PEI600/DNA complexes when they were pre-formed at an N/P ratio of 5.

types of complexes showed 2855-fold difference in gene expression level (Fig. 1B). We speculated that addition of PEI600 after NL4-10K might have blocked the interaction of the peptide with TrkA receptors. As such, the ‘polymer first’ method was used in the rest of the study.

3.2. Improving gene expression by NL4-10K: effects of the formation order of ternary complexes To investigate whether noncovalent, self-assembled ternary complexes prepared with our ‘polymer first’ method could be used to improve transfection efficiency, NIH3T3.E25, a stable cell line that constitutively expresses TrkA receptors, was tested for gene transfection mediated by PEI600/DNA/NL4-10K ternary complexes. Fig. 1B shows that with the increase in peptide concentration, transgene expression levels in E25 cells increased dosedependently and peaked at the N/P ratio of 5/1 between the peptide and DNA. Decrease of gene expression level was observed with the further increase in peptide concentration, probably because free, unbound NL4-10K competed with the ternary complexes for binding to TrkA receptors, thus reducing the cell uptake of plasmid DNA. To include more NL4-10K peptide into the ternary complexes so as to have better targeting effect, we formed the ternary complexes by mixing NL4-10K and DNA first and adding PEI600 later (the ‘peptide first’ method). We found that the change of formation order had no significant effect on the surface charge and particle size of the complexes (Table 1). However, this change had a profound effect on gene expression (Fig. 1B). While using the ternary complexes formed by the ‘polymer first’ method the transgene expression levels in E25 cells increased dosedependently; however, the change of peptide concentrations produced no effects at all when ternary complexes were prepared with ‘peptide’ first method. At the N/P ratio of 5/1/5 among PEI600, DNA and the peptide, the two

3.3. Specific gene delivery by PEI600/DNA/NL4-10K ternary complexes To examine the specificity of gene delivery by NL4-10Kcontaining ternary complexes, we included in the study a control peptide with 10-lysine sequence that is capable of binding to DNA but does not have the TrkA targeting moiety, in order to rule out the possibility that the improved gene expression by NL4-10K was due to a nonspecific effect of a positively charged peptide. We also extended our test to another TrkA-positive cell lines, rat PC12 cells. The change of peptides from NL4-10K to the control peptide 10K did not change surface charge and complex size significantly (Table 1). However, the change dramatically reduced gene expression to a level offered by PEI600/DNA complexes without peptides (Fig. 2A), suggesting a crucial effect of the targeting moiety of NL4-10K on a high level of gene expression provided by NL4-10K-containing ternary complexes. We also tested the effects of the increased amount of PEI600 on the NL4-10K ternary complex-mediated gene transfection in PC12 cells (Fig. 2A). At a high PEI600/ DNA N/P ratio of 10, while NL4-10K could still enhance transgene expression, background transfection by PEI600/ DNA complexes alone was also increased. Interestingly, at the PEI600/DNA N/P ratio of 20, we observed no enhancement of transgene expression by NL4-10K, indicating that excess PEI600 had blocked the binding of the targeting peptide to the PEI600/DNA complexes and that

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Fig. 1. NL4-10K binds to PEI600/DNA complexes and improves in vitro gene transfer in TrkA-positive cells: (A) DNA retardation by PEI600 and NL4-10K in agarose gel under electrophoresis. Plasmid DNA (0.2 mg) was complexed with PEI600 or PEI600 and NL4-10K at the indicated ratios and the complexes were electrophoresed in 0.7% agarose gel at 80 V for 50 min. (B) Effects of the formulation order of ternary complexes on transfection efficiency. Plasmid DNA was complexed either with NL410K followed by addition of PEI600 (the ‘peptide first’ method) or with PEI600 followed by addition of NL4-10K (the ‘polymer first’ method). The N/P ratio between PEI600 and DNA was 5/1 for all groups. The final N/P ratios listed as PEI600/DNA/NL4-10K are indicated. TrkA-positive NIH3T3.E25 cells in 24-well plates were transfected with complexes containing 1 mg of DNA for 4 h. Luciferase activity assays were preformed 1 day after cell transfection. The results are expressed in relative light units (RLU) per milligram of total cell protein (means7SD, n ¼ 4).

the targeting peptide binding was necessary for improved gene expression. To further examine the specificity of gene delivery by the NL4-10K-containing ternary complexes, we compared the transfection efficiency of the ternary complexes in two cell lines with stable TrkA expression, NIH3T3.TRK1 and NIH3T3.E25, with that in their parental cell line NIH3T3. The NL4-10K ternary complexes greatly increased gene expression in TrkA-expressing TRK1 and E25 cells, by 1148- and 548-fold, respectively, as compared with complexes with the control peptide 10K (Fig. 2B). However, these targeting complexes did not significantly affect gene expression in the NIH3T3 parental cells without TrkA (Fig. 2B). These results clearly demonstrate that the improved gene transfection by NL4-10K was related to the interaction of NL4 with TrkA receptors.

Fig. 2. Gene transfer specificity of the NL4-10K-containing ternary complexes: (A) gene expression mediated by NL4-10K-containing ternary complexes in TrkA-positive PC12 cells diminished when ternary complexes with an N/P ratio between PEI600 and DNA of 20 were used. In this experiment, the ratio between PEI600 and DNA increased from 5 to 20 and the N/P ratio between DNA and the peptide was fixed at 5. (B) Gene transfer in two TrkA-expressing NIH3T3 cell lines, TRK1 and E25, and their parental NIH3T3 cells. Complexes were prepared at an N/P ratio of 5/1/5 among PEI600, DNA and the peptide. Cells in 24-well plates were transfected with complexes containing 1 mg of DNA for 4 h. Luciferase activity assays were preformed 1 day after cell transfection. The results are expressed in relative light units (RLU) per milligram of total cell protein (means7SD, n ¼ 4).

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3.4. In vivo gene delivery by PEI600/DNA/NL4-10K ternary complexes We previously described the penetration of intrathecally applied gene delivery vectors to the dorsal root ganglion [24]. In view of the finding that a large portion of cells in the dorsal root ganglion expressed TrkA receptors [25], we used intrathecal injection in the rat in this study to test the in vivo efficiency of NL4-10K-containing ternary complexmediated gene delivery. After the injection, gene expression was observed both in the spinal cord and dorsal root ganglia (DRG). In the spinal cord, the gene expression level mediated by the NL4-10K-containing ternary complexes was similar to that provided the control complexes with the 10K peptide without the TrkA targeting moiety. In the DRG, there was a striking difference in gene expression levels provided by the two types of complexes, with the level from the NL4-10K-containing ternary complexes being 59-fold higher than that from the control complexes (Fig. 3A). This level of gene expression was close to those produced by widely used nonviral vectors PEI25k and lipofectamine (Fig. 3A). The difference between expression levels in the DRG and in the spinal cords indicates that transfection by NL4-10K-containing ternary complexes resulted in transgene expression preferably in the DRG, consistent with the targeting effect offered by NL4-10K peptide (Fig. 3B). This was remarkably different from transfection by PEI25k and lipofectamine, which mediated transgene expression equally significantly in both the DRG and the spinal cords (Fig. 3B). Immunostaining further confirmed the luciferase gene expression mediated by NL4-10K-containing ternary complexes in neurons of the dorsal root ganglion (Fig. 3C). 3.5. Biocompatibility of PEI600/DNA/NL4-10K ternary complexes Biocompatibility is one of the vital issues for successful gene therapy. Because of the introduction into our ternary complexes of the cationic polymer PEI600 that disrupts endosomal membranes and also because of the highly positively charged 10K domain, we investigated the possible in vitro cytotoxicity of PEI600/DNA/NL4-10K complexes in NIH3T3.E25 cells and primary rat cortical neurons. Continuous treatment for 48 h with PEI600/ DNA/NL4-10K complexes with an overall N/P ratio from 12.5 to 50 did not result in obvious cell death in both types of cells (Fig. 4). On the contrary, PEI25k/DNA complexes were apparently toxic to these cells. Exposure of the cells to PEI25k/DNA complexes, even at the lowest N/P ratio of 12.5, for 48 h led to the death of 80% of cells (Fig. 4). Zeta potential and particle size measurement indicated that PEI25k/DNA complexes at N/P ratios of 10 and 20 had slightly larger size and much higher values of surface charge than those of PEI600/DNA or PEI600/DNA/NL410K complexes at the same N/P ratios (Table 1).

Fig. 3. In vivo gene delivery to the dorsal root ganglia (DRG). NL4-10Kcontaining complexes (N/P ratio among PEI600/DNA/NL4-10K ¼ 5/1/5), and three control complexes, including the 10K peptide-containing ternary complexes (N/P ratio among PEI600/DNA/10K ¼ 5/1/5), PEI25k complexes (N/P ratio between polymer/DNA ¼ 10/1), and lipofectamine complexes (lipid/DNA ratio ¼ 3/1), were injected intrathecally into the lumbar spinal cord in rats. The DRG and spinal cords were collected 2 days after injection. The results are expressed in relative light units (RLU) per milligram of total cell protein in (A) and RLU per milligram of protein in the DRG as % of the luciferase activities in the spinal cord in (B). Five rats were used for each group and the error bars represent standard deviation. (C) Confocal images show the luciferase expression in neurons of the DRG. Frozen sections of rat DRG were collected 2 days after injection of NL4-10K-containing ternary complexes (N/P ratio among PEI600/DNA/peptide ¼ 5/1/5) and used for double immunostaining against neuron-specific nuclear protein (NeuN) to show neurons and against luciferase protein (Luc) to show transfected cells.

By using a microglial activation marker OX42 to detect inflammatory responses that could be triggered directly by gene delivery vectors or indirectly by cell/tissue injury [26], the nervous tissue compatibility of these DNA complexes was assayed in vivo in the rat spinal cord. PEI600/DNA/ NL4-10K (10/1/5) complexes showed no obvious effects on

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Fig. 4. In vitro cytotoxicity of NL4-10K-containing ternary complexes and PEI25k/DNA complexes in NIH3T3.E25 cells (A) and primary rat neurons (B). The ternary complexes were prepared by mixing PEI600 with DNA at an N/P ratio of 5 or 10 first for NIH3T3.E25 and primary cortical neurons, respectively, followed by addition of various amounts of NL410K to the indicated N/P ratios. PEI25k/DNA complexes at the indicated N/P ratios were used as positive controls. The complexes were added at 0.1 mg DNA per well in 96-well plates. Cells were incubated with the complexes for 48 h before being analyzed in a MTT assay. Cell viability was expressed as percentage of control, i.e. the untreated sister cultures. Each point represents the mean7SD of four cultures.

OX42 immunostaining in the spinal cord region close to the injection site, while the PEI25k/DNA complexes (15/1) induced a clear inflammatory response, as demonstrated by the proliferation of OX42-stained microglial cells (Fig. 5). 4. Discussion Covalent conjugation of targeting moieties with gene delivery vectors requires chemical reactions, which could be detrimental to the bioactivities of sensitive moieties. In the case of using proteins or peptides that are difficult to be purified or synthesized, the relatively large amounts of materials required for chemical conjugation could be prohibitively expensive and limit the scale-up of vector preparation necessary for human gene therapy application. During the preparation of the ternary complexes used in the current study, targeting peptides were introduced simply through electrostatic interaction between the positively charged DNA-binding sequence of the peptides and negatively charged DNA. This straightforward selfassembly method circumvents the need of aggressive and reagent-wasteful chemical reactions and would preserve the bioactivities of peptide-based targeting moieties more

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Fig. 5. In vivo biocompatibility of NL4-10K-containing ternary complexes. Immunoreactivity of OX42 in the spinal cord after intrathecal injection of NL4-10K-containing ternary complexes was shown. PEI600 was mixed with DNA at an N/P ratio of 10, and NL4-10K was then added at an N/P ratio of 5 to form ternary complexes. PEI25k/DNA complexes formed with an N/P ratio of 15 were used as positive control. Complexes containing 3 mg DNA in a volume of 50 ml were injected intrathecally. A control group injected with a 5% glucose solution was included as negative control. Samples were collected 3 days after injection and the tissue sections were stained with anti-OX42, followed by immunoperoxidase histochemistry. Note the increase in the number of OX42-stained microglial cells after injection of PEI25k/DNA complexes.

effectively. In addition to cell targeting, peptides with other biological functions can be incorporated into the complexes for other gene delivery purposes, such as endosomolysis or nuclear transport. PEIs with MW of 25,000 Da or greater have shown high transfection efficiency both in vitro and in vivo [20,27]. These higher MW cationic polymers, however, induce rapid necrotic-like changes resulting from perturbation of the plasma membrane and the activation of a mitochondrially mediated apoptosis [28]. Low MW PEIs (o2000 Da) displayed much less toxicity but almost no transfection activity [29]. Thus, PEI600 was employed in the current study not just because of its endosomolytic function, but also because of its lower cytotoxicity and lower background transfection efficiency. The excess positive charge on polymer/DNA complexes can cause nonspecific binding and uptake by nontargeted cells, thereby reducing the targeting efficiency of delivery vectors [30]. We prepared PEI600/DNA complexes at an

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N/P ratio of 5 that displayed a weak positive surface charge to facilitate the interaction of the complexes with the targeting NL4-10K peptide. This had proved to be crucial for the specific gene delivery by our ternary complexes. The use of PEI600 polymers with an N/P ratio less than 5 failed to form stable polymer/DNA complexes. A ratio higher than 5 led to an increase of nonspecific cellular uptake of the complexes, most likely due to the failure to incorporate enough targeting moieties to the complexes caused by charge repelling between PEI600 and the 10K domain of our peptides. After addition of the targeting peptide at the N/P ratio of 5, the surface charge of the ternary complexes reached 23 mV. This is still significantly lower than that of PEI25k/DNA complexes at the same N/P ratio, which was about 42 mV. A weakly positively charged surface might be beneficial to the specific binding of targeting ligands. Moreover, DNA delivery complexes with such a surface charge should have less interaction with blood components and the negatively charged cell surface, thus probably exhibiting less stimulation of the host immune system and clearance mechanism. We found that the formation order to make our peptide/ polymer/DNA ternary complexes had an enormous effect on gene delivery efficiency in the current study. While the complexes prepared by packing DNA with PEI600 first and adding NL4-10K afterward (the ‘polymer first’ method) increased gene expression significantly, the complexes generated vise versa (the ‘peptide first’ method) only produced transgene expression at the same level as that of the control complexes. Noticeably, the surface charges and particle sizes of the complexes prepared by these two different ways were similar. It is possible that the ‘polymer first’ method resulted in a ternary structure with PEI600 and 10K domain bound to the DNA in the core of the complexes, while the loop region of NL4, the TrkA targeting domain, was on the surface of the complexes due to the b-strands of the NL4 hairpin motif in the chimeric peptide that act as spacers between the NL4 loop region and the 10K DNA binding domain. Therefore, the complexes formed by the ‘polymer first’ method could be easily taken up into the TrkA-positive cells. In the ‘peptide first’ method, however, the NL4 loop region might have been masked by PEI600 that was added subsequently. Due to the complexity of biological system, in vivo targeted gene transfer is challenging. In the current study, introducing NGF receptor targeting peptides to PEI600/ DNA complexes had influenced the distribution of the complexes towards a region expressing TrkA receptors, thus gene expression was observed preferably in the DRG. In contrast, intrathecally injected PEI25k and lipofectamine complexes, two delivery systems without targeting ligands, mediated gene transfer with equal effectiveness in the regions with and without TrkA receptors. Thus, our targeted delivery system might have useful clinical applications. One of such applications is to prevent nerve degeneration in peripheral neuropathies through therapeutic gene delivery to neurons in the dorsal root ganglion [24].

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