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Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

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Research Paper

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Evaluation of improved PAMAM-G5 conjugates for gene delivery targeted to the transferrin receptor

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Koldo Urbiola a, Laura Blanco-Fernández a, Gemma Navarro b, Wolfgang Rödl c, Ernst Wagner c, Manfred Ogris d, Conchita Tros de Ilarduya a,⇑ a

Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, 31080 Pamplona, Spain Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA 02115, USA Pharmaceutical Biotechnology, Center for NanoScience (CeNS), Ludwig-Maximilians-University (LMU) Munich, Germany d Center of Pharmaceutical Sciences, University of Vienna, Austria b c

a r t i c l e

i n f o

Article history: Received 13 November 2014 Revised 6 March 2015 Accepted in revised form 7 May 2015 Available online xxxx Keywords: Gene therapy Gene transfer Transferrin Nanoparticle Polyamidoamine (PAMAM) dendrimers Nanotechnology

a b s t r a c t The transfection activity of non-viral vectors is highly dependent on the delivery capacity of the carriers. Therefore, the aim of this work was to evaluate the activity of a new PAMAM dendrimer-Transferrin conjugate (P-Tf) with improved gene delivery activity to cancer cells. The formulations containing the novel P-Tf were able to bind pDNA and protect it from the activity of DNAse I enzyme. Moreover, it formed nanoparticles with positive surface charge, although the presence of Tf led to a decrease of the zeta potential to almost electroneutral values. This new vector, formulated at N/P 6, exhibited excellent transfection efficacy in HeLa, HepG2 and CT26 cell lines, whereas in Neuro2A no improvement was achieved. Compared to control complexes with branched polyethylenimine (bPEI), targeted dendriplexes (complexes formed by cationic polymeric dendrimers and DNA) were more efficient in HepG2 and HeLa cells. Cellular viability was always kept over 80% in these cell lines with higher values than bPEI control polyplexes. The uptake via receptor-mediated endocytosis was ensured by a competition assay, by adding an excess of free Tf, which led to a decrease in the transfection activity of targeted dendriplexes. Ó 2015 Elsevier B.V. All rights reserved.

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1. Introduction

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The use of strategies to improve the activity of non-viral vectors for gene delivery has been one of the most important issues to be overcome in order to enhance the application of these systems to clinical practice. One major approach in non-viral gene therapy is based on cationic polymers, such as polyethyleneimine (PEI), polylysine (PLL), polyamidoamine (PAMAM) or polypropyleneimine (PPI) [1,2]. This kind of vectors is interesting since they are easily manufactured, flexible, versatile and are able to mediate the delivery of natural or synthetic nucleic acid of any kind and size [2–4]. As this type of vectors has difficulties in obtaining high levels of expression, especially in the presence of serum, some of the most used strategies have included the addition of anionic molecules in order to shield the surface charge, inclusion of new components such as penetrating peptides, activation of the structure of dendrimers and targeting nanosystems to specific receptors [5–7]. Regarding the last approach, one of the most used receptors for the delivery of drugs and improving the activity of non-viral

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⇑ Corresponding author. Tel.: +34 948/425600x80 6375. E-mail address: [email protected] (C. Tros de Ilarduya).

vectors is the transferrin receptor (TfR), which has been reported to be overexpressed on the surface of different cancer cells [8,9]. This receptor is the principal route by which iron is internalized into cells. It is considered an ubiquitous receptor expressed on most active proliferating cell types and its expression is upregulated in multiple cancer cells since iron is a basic element during the DNA synthesis, cell division and cellular metabolism [10–12]. Its ligand, transferrin (Tf), presents a molecular weight of 80 kDa and it is one of the proteins in charge of the iron transport through the body [11]. The uptake of Tf via TfR involves a clathrin dependent endocytosis process that leads to two different intracellular pathways. One involves the recycling of the complex Tf-TfR and the other one the lysosomal degradation. It is believed that only 5–15% of the intracellular pathways follow the lysosomal and subsequent elimination route [13]. Concerning the use of Tf to enhance gene delivery, in 1990, Wagner et al. [14] introduced the term ‘‘transferrinfection’’ to define the transfection procedure when Tf is included as a ligand to enhance the uptake of DNA. Since then, the use of Tf as a ligand to enhance the delivery of drug conjugates or nanoparticles has increased its interest and has proven to be effective, not only for the enhancement in gene delivery and expression, but also for

http://dx.doi.org/10.1016/j.ejpb.2015.05.007 0939-6411/Ó 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: K. Urbiola et al., Evaluation of improved PAMAM-G5 conjugates for gene delivery targeted to the transferrin receptor, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.05.007

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the potential use as a shielding and targeting agent of polyethyleneimine containing polyplexes and other cationic polymers after the administration in vitro and in vivo in tumor-bearing mice [14–19]. In parallel, Xu et al. described the administration of Tf-liposomes carrying the p53 gene that result in the regression of a human neck and head cancer model [20]. Tros de Ilarduya et al. published a study where Tf, electrostatically assembled, was able to enhance the transfection activity of cationic liposomes in vitro and in vivo [21] and the ability of that formulation to produce the regression of a CT26 tumor after the administration of the interleukin 12 gene [22]. This ligand has also exhibited good activity in enhancing the delivery of siRNA as Cardoso et al. reported for Tf-lipoplexes [23,24]. PAMAM dendrimers, described in 1986 by Tomalia et al. [25] present exceptional chemical features for the modification of the surface groups and have been used as imaging agents or drug carriers [26,27]. The ability to target the Tf receptor by these systems has been studied by Li et al. [28], who designed targeted PAMAM-G4 dendrimers with Tf carrying tamoxifen that were able to mediate an effective transport across the blood–brain barrier and induce the inhibition and death of glioma cells. The ability of the PAMAM dendrimers to mediate an effective gene transfer has also been demonstrated [27,29] but their use as DNA or RNA carriers targeted to the Tf receptor has not been widely examined. In this respect, Huang et al. [30,31] studied the possibility of targeting brain tissue through the use of lactoferrin-PAMAM and transferrin-PAMAM conjugates, suggesting that the proposed dendriplexes can be exploited as potential non-viral gene vectors targeting the brain via non-invasive administration. Therefore, the aim of this study was to evaluate the transfection activity of a new Tf containing dendriplex for gene delivery. We propose a novel PAMAM-Tf conjugate for gene delivery as a new carrier for pDNA delivery that can improve the transfection efficacy of the dendriplexes used so far and enhance the activity of the commercial available PAMAM by attaching the targeting ligand transferrin to the formulation.

was hydrated with 0.25 M sodium chloride and 20 mM HEPES. A solution of 625 nmol of human transferrin (hTf) dissolved in 30 mM sodium acetate buffer (pH 5) was cooled to 0 °C and three molar equivalents of sodium periodate in 30 mM sodium acetate buffer were added. The mixture was kept on ice for 90 min. For removal of the low molecular weight products, gel filtration (Sephadex G-25 superfine, 30 mM sodium acetate buffer pH 5) was performed (monitoring: UV absorption at 280 nm) resulting in 424 nmol oxidized hTf. The modified transferrin solution was added to 508 nmol of PAMAM G5 in 0.25 M sodium chloride, 20 mM HEPES buffer and vigorously mixed at room temperature. The pH was adjusted to 7.3 by the addition of 2 M HEPES pH 7.9. After 30 min, four portions of sodium cyanoborohydride (1 mg per 10 mg transferrin) were added at 1 h intervals. After 19 h, the salt concentration was adjusted to 0.5 M by addition of 3 M sodium chloride. The mixture was loaded on to cation-exchange column (MacroPrep High S 10/10, Bio-Rad, Hercules, CA, USA) and fractioned with a salt gradient from 0.5 M to 3 M sodium chloride (with a constant content of 20 mM HEPES pH 7.3). The major amount of conjugate eluted between 2.1 and 3 M salt. After dialysis against 4 L of HBS (20 mM HEPES, 150 mM NaCl, pH 7.4), the conjugate (designated P-Tf) was obtained at a molar ratio of transferrin: PAMAM-G5 of 1:0.94. TNBS (trinitrobenzene sulfonate) assay for determination of primary amines in PAMAM G5 was performed. The amount of transferrin was determined by absorption measurement at 280 nm. Iron was incorporated by the addition of 1.25 lL 10 mM iron(III) citrate buffer (200 mM citrate, pH 7.3 adjusted with sodium bicarbonate) per milligram of transferrin. The chemical synthesis of the PAMAM-Transferrin (P-Tf) conjugate was performed in analogous way as for the coupling of hTf to BPEI [18] by Schiff-base formation between primary amines in PAMAM and oxidized sugar residues in hTf followed by reductive amination. This process was always performed at room temperature; therefore the differences generated during the subsequent experiments using P-Tf conjugates have to be related with the presence of transferrin and are not likely to be due to different processes that can increase the activity of PAMAM dendrimers, e.g. activation of the structure.

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2.3. Preparation of complexes

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2. Materials and methods

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2.1. Materials

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First, methyl alcohol was removed from the commercial PAMAM solution by rotary evaporation under reduced pressure. Then, the film was hydrated with Buffer HEPES (10 mM) Glucose 10% w/v (pH 7.4) (BHG) to a final concentration of 0.25 mg/mL. Plain dendriplexes were formed by incubating equal volumes of pDNA and dendrimers at room temperature (20–25 °C) for 15 min. Complexes were formulated at different N/P ratios, which represented the molar ratio of positively charged primary amines in PAMAM and the phosphate groups in the pDNA backbone. Control polyplexes formulated with bPEI (N/P 6) were formed using the same protocol. Targeted dendriplexes were prepared similar to plain dendriplexes but an amount of commercial PAMAM was substituted by the P-Tf conjugate. This means that, commercial PAMAM G5 was partially replaced by P-Tf and the mixture was condensed with pDNA at different N/P ratios. The degree of replacement is represented as percentage (e.g. 25% means 1 part of P-Tf plus 3 parts of commercial PAMAM).

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Polyamidoamine dendrimer generation 5 (PAMAM) with ethylenediamine core (MW 28,825 Da, 128 N-terminal amines), branched polyethylenimine (25 kDa) and human apo-transferrin, were purchased from Sigma–Aldrich. The plasmid pCMVLuc (BioServe Technologies, Maryland, USA) encoding luciferase gene was used in the transfection studies. Plasmid was amplified in E. coli, isolated and purified using a QUIAGEN Plasmid Giga Kit (QUIAGEN, Germany). DNA concentration and purity were measured by NanoDrop (NanoDrop ND1000, Thermo Scientific). Agarose, TRIS, boric acid and ethylenediaminetetraacetic acid (EDTA) were provided by Sigma Aldrich (Barcelona, Spain). The HEPES glucose buffer (pH 7.4) was prepared from D-(+)-glucose and N-(2-hydroxyethyl) piperazine-N0 -[2-ethanesulfonic acid] (HEPES, Sigma–Aldrich). Alamar Blue Dye was purchased from Invitrogen (Barcelona, Spain), and was used in the toxicity studies. Human transferrin for competition assay was purchased by BD Biosciences (Bedford, Massachusetts, USA).

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2.2. Synthesis of Transferrin-PAMAM conjugates

2.4. Gel retardation studies

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Transferrin-PAMAM conjugate synthesis was performed similarly as described previously for Tf-PEI (25 kDa) [18]. Firstly, methyl alcohol was removed from the commercial PAMAM solution by rotary evaporation under reduced pressure. Then, the film

For gel retardation studies, complexes containing 1 lg of DNA prepared at different N/P ratios in BHG were electrophoresed through a 0.8% agarose gel using TBE buffer (100 mM TRIS, 90 mM boric acid, 1 mM EDTA, pH 8.4). The gel was stained with

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ethidium bromide, electrophoresed for 1 h, 100 mV and visualized under UV illumination (Doc 2000, Bio-Rad, USA).

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2.5. DNAse I protection assay

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Dendriplexes were prepared at different N/P ratios containing different percentages of P-Tf at a final concentration of 100 lg of pDNA/mL in a total volume of 20 lL. After the formation of the complexes, DNAse I (1 U/lg pDNA) was added to each sample and the mixtures were incubated at 37 °C for 1 h. In order to stop the DNAse I activity and disassemble the dendriplexes, 5 lL of EDTA 0.25 M and 5 lL of SDS (10% w/v in water) were added. Then, the samples were electrophoresed as described in the gel retardation studies. Non-treated and digested pDNA were included as controls.

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2.6. Size and zeta potential determination

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Particle size was measured by Dynamic Light Scattering (DLS) and the overall charge by zeta-potential measurements, using a particle analyzer (Zeta Nano Series; Malvern Instruments, Barcelona, Spain). For the electrophoretic mobility determinations, samples were prepared with Buffer Hepes (10 mM) glucose 10% (w/v) pH 7.4 at a final concentration of 10 lg pDNA/mL. Experimental conditions were as follows: Tª (25 °C), dispersant refractive index 1.33 (water), viscosity (0.9 cP), dispersant dielectric constant: 78.5.

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2.9. Toxicity studies

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Cell viability was quantified by Alamar Blue assay. 100,000 cells per well were seeded and grown overnight in 48 well culture plates. Cells were transfected as described previously. After 4 h complexes were removed and substituted by new fresh media. 48 h later, media were removed and 1 mL of 10% (v/v) Alamar Blue dye in medium supplemented with 10% FBS was added to each well. After 2.5 h of incubation at 37 °C, 200 lL of the supernatant was assayed by measuring the absorbance at 570 and 600 nm. Cell viability was calculated according to the following formula:

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ðA570  A600 Þtreated cells % Viability ¼ 100  ðA570  A600 Þcontrol cells

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2.10. Statistical analysis

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Results are reported as the mean values ± standard deviation. Statistical analysis was performed with SPSS 15.0 (SPSSÒ, Chicago, IL, USA). The different transfection activities in vitro were compared with ANOVA (Tukey post hoc adjust). Differences were considered statistically significant at p < 0.05.

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3. Results and discussion

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2.7. Cell culture

3.1. Electrostatic interaction of pDNA and P-Tf

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The formation of complexes and the binding of dendrimers to DNA were ensured by examining the retardation in the migration of the plasmid DNA after agarose gel electrophoresis. Fig. 1 shows that different N/P ratios and percentages of P-Tf incubated with 1 lg of plasmid DNA resulted in a total electrophoretic immobilization of DNA in all the cases, showing the strong electrostatic interaction of pDNA with dendrimer molecules.

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HepG2 (human hepatoblastoma), HeLa (human cervix carcinoma), Neuro2A (murine neuroblastoma) and CT26 (murine colon carcinoma) cell lines were obtained from the American Type Culture Collection (Rockville, Maryland). Cell lines were maintained at 37 °C under 5% CO2 in Dulbecco’s modified Eagle’s medium high glucose, supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and penicillin (100 units/mL), streptomycin (100 lg/mL) and L-glutamine (4 mM) (Gibco BRL Life Technologies, Barcelona, Spain). Cells were trypsinized twice a week.

3.2. Plasmid protection inside the dendriplexes

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2.8. In vitro gene transfer

To ensure the ability of targeted PAMAM-dendriplexes to protect the complexed pDNA, these vectors were exposed to DNAse I. Fig. 2 shows the stability of the complexes in serum, given that free plasmid is completely degraded by the enzyme (lane 2) whereas plasmid complexed with dendrimers appears intact after the dissociation of the dendriplexes (Lanes 3–14). These data provide an interesting basis for considering the P-Tf conjugate as a promising non-viral gene delivery carrier in vivo since, once put into circulation, vectors are subjected to serum inactivation and enzymatic degradation of the complexes, causing the loss and disappearance of the pDNA and the subsequent effect [32,33].

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For each transfection, 100,000 cells were seeded in 1 mL of medium in 48-well culture plates (Iwaki, Japan) and incubated for 24 h at 37 °C in 5% CO2. The primary growth medium was removed and replaced with 0.3 mL of new media and 0.2 mL of complexes formulated in BHG containing 1 lg of pDNA per well. Transfections were done in the presence of 10% FBS. After 4 h of incubation, complexes were removed and replaced with cell culture medium containing 10% FBS. 48 h later, cells were washed with phosphate-buffered saline and lysed using 100 lL of Reporter Lysis Buffer (Promega, Madison, Wisconsin, USA) at room temperature for 10 min, followed by two freeze–thaw cycles. The cell lysate was centrifuged for 2 min at 12,000g to pellet debris. 20 lL of the supernatant was assayed for total luciferase activity using the Luciferase Assay Reagent (Promega), according to the manufacturer’s protocol. A luminometer (Sirius-2; Berthold Detection Systems, Innogenetics, Diagnóstica y Terapéutica, Barcelona, Spain) was used to measure luciferase activity. The Bio-Rad Dc Protein Assay (Bio-Rad Laboratories, USA), using bovine serum albumin as the standard, was used for quantifying protein content. Data were expressed as nanograms of luciferase (based on a standard curve for luciferase activity) per milligram of protein.

Fig. 1. Retardation assay of plain and targeted nanoparticles containing increasing percentages of P-Tf (0%, 16%, 25% and 50%) at different N/P ratios: N/P 2 (lanes 2, 3, 4, 5), N/P 4 (lanes 6, 7, 8, 9), and N/P 6 lanes (10, 11, 12, 13). DNA was included as control (lane 1).

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K. Urbiola et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2015) xxx–xxx Table 1 Particle size (diameter) and surface charge at different N/P ratios and increasing percentages of P-Tf (0%, 16%, 25% and 50%). The data are represented as the mean ± s.d. of three independent measurements. Polydispersion index (PDI).

Fig. 2. Protection assay. Untreated DNA (lane 1), DNA treated with DNAse I (lane 2). Dendriplexes containing increasing percentages of P-Tf (0%, 16%, 25% and 50%) at different N/P ratios: N/P 2 (lanes 3, 4, 5, 6), N/P 4 (lanes 7, 8, 9, 10), and N/P 6 (lanes 11, 12, 13, 14).

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3.3. Characterization of particle size and zeta potential

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Particle size and surface charge were characterized in order to study the influence of both, the N/P ratio and the presence of the ligand transferrin (Table 1). The DLS measurements showed particles in the nanometric range, with a relatively low polydispersity index (PDI) values in all the cases, independently of the percentage of P-Tf included and the N/P ratio. A tendency toward smaller particle size as the N/P ratio increases was shown, maintaining optimal hydrodynamic diameters under 150 nm. The surface charge was positive for all the conditions assayed but the presence of increasing percentages of P-Tf did produce a significant decrease of the zeta potential to almost electroneutral values for N/P 2 and 4 in the case of 50% P-Tf containing complexes (3.3 and 2.2 mV respectively). Zeta potential values did also exhibit an increase as the N/P ratio grew. Increasing the N/P ratio produced a drop in the particle size. The addition of increasing percentages of P-Tf led to a decrease of the surface charge, down to almost electroneutral zeta potential values, in accordance with the anionic structure and charge shielding properties of the protein which was also observed in previous studies [18,21]. It is known that electroneutral values may generate an increase in particle size since the absence of charge repulsion leads to the formation of bigger aggregates. This relation between size and surface charge has been described for liposomes [34,35]. Nevertheless, our data point out that, despite presenting a slight increase in particle size when the highest amount of P-Tf is included, stable nanoparticles are formed in terms of size and zeta potential, even at low N/P ratios without presenting important differences between targeted dendriplexes and non-targeted ones. Therefore, the subsequent variations of the transfection activity of these dendriplexes should be related to the presence of Tf and cannot be attributed to unequal sizes of the formulations. These data are in agreement with the presented results of protection against DNAse I and pDNA immobilization (Figs. 1 and 2).

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3.4. In vitro transfection activity

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Transfection activity of PAMAM/P-Tf/pDNA dendriplexes was evaluated by using the cell lines HeLa, HepG2, Neuro2A and CT26 (Fig. 3). The behavior of the dendriplexes in CT26 and HeLa cells showed a tendency to increase luciferase activity when the percentage of P-Tf increased at any N/P ratio. In CT26 cells at N/P 6, the dendriplexes containing 50% of P-Tf produced the highest luciferase expression, which resulted in 32.5 times higher activity compared to plain dendriplexes (p < 0.001). In HeLa cells, the substitution of 50% P-Tf in the formulations did produce a statistically significant rise of the luciferase activity compared to the plain dendriplexes at N/P ratios 4 and 6 (p < 0.001). The use of 50% P-Tf within the formulation at N/P 6 ratio exhibited a 5.4-fold increase in the luciferase values (p < 0.001) in HepG2 cells compared to plain dendriplexes. The substitution of commercial PAMAM by P-Tf did not result in any increment at N/P 2 and 4.

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Zeta potential (mV)

Conductivity (mS/cm)

N/P ratio

%P-Tf

Size (nm)

PDI

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0 16 25 50

108.0 ± 0.8 106.2 ± 0.6 109.1 ± 1.0 114.3 ± 0.5

0.25 0.32 0.25 0.23

8.0 ± 0.2 6.7 ± 1.6 6.5 ± 0.0 3.3 ± 0.1

0.3 ± 0.1 0.5 ± 0.1 1.1 ± 0.2 1.7 ± 0.1

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0 16 25 50

84.3 ± 1.3 95.2 ± 0.4 105.5 ± 0.6 145.0 ± 1.6

0.27 0.18 0.18 0.25

17.5 ± 1.3 8.3 ± 0.9 5.8 ± 1.1 2.2 ± 0.1

0.4 ± 0.1 0.6 ± 0.2 0.9 ± 0.2 1.6 ± 0.1

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0 16 25 50

80.4 ± 0.9 83.1 ± 0.5 80.9 ± 0.2 85.1 ± 1.6

0.24 0.25 0.22 0.23

22.1 ± 0.0 11.0 ± 0.0 11.1 ± 0.8 8.1 ± 0.6

0.3 ± 0.1 0.5 ± 0.1 1.0 ± 0.2 1.8 ± 0.1

The transfection with P-Tf in Neuro2A cells did not show any improvement independently of the percentage of P-Tf and the transfection activity was always significantly reduced. The increment in the N/P ratio led to an increase in the transfection activity for each degree of substitution with P-Tf. Compared to control bPEI-polyplexes, PAMAM dendriplexes showed higher gene expression values in HeLa, HepG2 and Neuro2A cells. In contrast, in the CT26 cell line, control polyplexes achieved higher transfection activity than dendriplexes. The inclusion of anionic molecules, usually, in order to shield the surface charge, has been reported to produce complexes with lower transfection activity, since the drop of the surface charge generates a blockage in the non-specific adsorptive endocytosis [36,37]. The absence of improvement in the HepG2 cell line at N/P 2 and 4 could also be related to the TfR data reported by Sakaguchi et al. [38], who claims that, apparently, the expression of the TfR is low for HepG2 cells. Moreover, the comparison of the TfR internalization results between different cell lines suggested that this cell line presents low internalization values, which was considered an indicative of low activity of the receptor mediated endocytosis. This implies that a large fraction of the TfR is trapped in early endosomes of a static nature. Hence, it may be possible that, with the lowest N/P ratios, 2 and 4, transfection could not be carried out as the presence of Tf could not be enough for enhancing luciferase expression and higher zeta potential values might be needed for an effective gene transfer. HeLa cells, considered a positive cell line for TfR [38], presented a better transfection pattern for N/P 4 and 6 and a statistically significant increment was described when 50% of commercial PAMAM was substituted by P-Tf conjugate. Between both ratios, differences could not be described and similar values were achieved. N/P 6 ratio was selected as the best condition in order to perform the subsequent competition studies. In contrast to previous results, where Neuro2A, a TfR positive cell line [39], is used as a model with TfR [40], this cell line did not show any improvement by using P-Tf conjugates. The reason why this cell line presents different expression patterns for the same complexes could be related to the fact that transfection processes are highly dependent on the cell line and the complex used [41]. Apart from that, cell lines will differ not only in their level of TfR, but also in the presence of glycosaminoglycans, such as heparan sulfate, which can contribute to mask the transferrin-mediated gene transfer by the adsorptive endocytosis. Moreover, another possible reason is that the large Tf molecule may be hampering endosomal release, which could be more prominent in the Neuro2A cells than in the other cell lines tested.

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Fig. 3. In vitro transfection activity by plain and targeted dendriplexes containing increasing percentages of P-Tf in CT26, HeLa, HepG2 and Neuro2A in the presence of 10% FBS. Plain bPEI polyplexes were used as control. Data represent the mean ± s.d. and are representative of three independent experiments.

Fig. 4. Competition assay by adding an excess of free Tf (5 mg/mL) to HeLa cells previous to transfection of plain and targeted dendriplexes at N/P 6.

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3.5. Uptake mechanism

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In order to clarify whether the uptake mechanism of P-Tf conjugates was mediated via receptor-mediated endocytosis, as described for other targeted complexes [40] a competitive inhibition experiment was performed in HeLa cells. Transfection activity

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in the presence of an excess of free Tf (5 mg Tf/mL) showed a decrease of the luciferase expression values when commercial PAMAM had been substituted by P-Tf, independently of the percentage of substitution (p < 0.05). Non-targeted dendriplexes showed similar transfection values after blocking the transferrin receptor (Fig. 4). The result pointed out that the transfection activity is inhibited by the presence of free Tf in the transfection medium, which can produce a saturation and subsequent reduction of the transfection efficacy by blocking the Tf-receptor.

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3.6. Toxicity studies

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The toxicity of the new P-Tf conjugate had not been previously studied. Alamar Blue dye was used to verify it. The result confirmed that plain PAMAM dendriplexes and the mixtures with P-Tf did not exhibit toxicity in the conditions assayed for HeLa, CT26 and HepG2 and viability values were always above 80% (p < 0.001) (Fig. 5). For Neuro2A, cell viability dropped down to 60%. Compared to bPEI polyplexes, plain and targeted dendriplexes were less toxic in all the conditions assayed. The absence of acute toxicity supports the use of P-Tf conjugate and suggests that the differences found in the transfection activity studies must be related to the presence of TfR and the P-Tf conjugate, which is supposed to enhance the expression of the transgene, and not with a different viability of the cells.

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Fig. 5. In vitro cellular viability by plain (non-targeted) and targeted dendriplexes containing increasing percentages of P-Tf in CT26, HeLa, HepG2 and Neuro2A in the presence of 10% FBS. Plain bPEI polyplexes and non-treated cells were used as control. Data represent the mean ± s.d. of three wells and are representative of three independent experiments. 432

4. Conclusions

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In this work we have evaluated the ability of a new PAMAM-Transferrin conjugate to form nanoparticles in the presence of pDNA and its transfection activity. The results showed that the dendriplexes containing the novel P-Tf conjugate were able to form stable particles and protect the complexed pDNA. The particles showed nanometric size with positive surface charge, although the inclusion of Tf led to a decrease of the zeta potential values. The presence of P-Tf in the complexes formulated at N/P 6 produced a statistically significant increase in the transfection activity when 50% of P-Tf was included in CT26, HeLa and HepG2 cell lines, whereas in Neuro2A the substitution of 50% of commercial PAMAM by P-Tf always led to a decrease in the gene expression. Moreover, the competition assay confirms that the uptake is mediated by specific receptor-mediated endocytosis. Targeted and plain dendriplexes exhibited low toxicity, except for Neuro2A cell line, where cell viability fell down to 60%. The toxicity of P-Tf dendriplexes was found to be lower compared to that of bPEI-polyplexes, frequently used as non-viral gene delivery systems. In our case, we have formulated dendriplexes for targeted gene transfer with increased and improved transfection activity to the transferrin receptor (TfR).

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Acknowledgments

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This work was financially supported by the Government of Navarra (Department of Innovation and Industry) (Ref.

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IIQ14334.RI1), the FEDER fundings from the European Commission and the University of Navarra Foundation (FUN). We also acknowledge Ainhoa Algaba for her help with the competition experiment and the ‘‘Asociación de Amigos de la Universidad de Navarra’’ for the fellowship to Koldo Urbiola.

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