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Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps 5 6 3 4 7 8 9 10 11 12 13 14 15 1 3 7 5 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

Synthesis and characterization of a PAMAM dendrimer nanocarrier functionalized by SRL peptide for targeted gene delivery to the brain Amir Zarebkohan a, Farhood Najafi b, Hamid Reza Moghimi c, Mohammad Hemmati a, Mohammad Reza Deevband a, Bahram Kazemi d,e,⇑ a

Biomedical Engineering and Medical Physics Department, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Department of Resin and Additives, Institute for Color Science and Technology, Tehran, Iran School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran d Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran e Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran b c

a r t i c l e

i n f o

Article history: Received 10 April 2015 Received in revised form 17 June 2015 Accepted 25 June 2015 Available online xxxx Chemical compounds: Phenylarsine oxide (PubChem CID: 4778) Ethylenediamine (PubChem CID: 3301) Methyl acrylate (PubChem CID: 4093) Filipin complex (PubChem CID: 6433553) L-Polylysin (PubChem CID: 162282) NHS-PEG-MAL (PubChem CID: 51340913) BODIPY fluorophore (PubChem CID: 13007361) NaHCO3 (PubChem CID: 767) Agarose (PubChem CID: 11966311) DMSO (PubChem CID: 679) Colchicines (PubChem CID: 6167) Ethidiummonoazide bromide (PubChem CID: 2762649) TE buffer (PubChem CID: 16218629) Sodium sulfate (PubChem CID: 3423265) MTT (PubChem CID: 64965) Formazin (PubChem CID: 9567750) DAPI (PubChem CID: 2954) Diethuyl ether (PubChem CID: 3283) Paraformaldehyde (PubChem CID: 712) Sucrose (PubChem CID: 5988)

a b s t r a c t Blood–brain barrier inhibits most of drugs and genetic materials from reaching the brain. So, developing high efficiency carriers for gene and drug delivery to the brain, is the challenging area in pharmaceutical sciences. This investigation aimed to target DNA to brain using Serine–Arginine–Leucine (SRL) functionalized PAMAM dendrimers as a novel gene delivery system. The SRL peptide was linked on G4 PAMAM dendrimers using bifunctional PEG. DNA was then loaded in these functionalized nanoparticles and their physicochemical properties and cellular uptake/distribution evaluated by AFM, NMR, FTIR and fluorescence and confocal microscopy. Also, biodistribution and brain localization of nanoparticles were studied after IV injection of nanoparticles into rat tail. Unmodified nanoparticles were used as control in all evaluations. In vitro studies showed that SRL-modified nanoparticles have good transfection efficacy and low toxicity. Results also showed that SRL is a LRP ligand and SRL-modified nanoparticles internalized by clathrin/caveolin energy-dependent endocytosis to brain capillary endothelial cells. After intravenous administration, the SRL-modified nanoparticles were able to cross the blood–brain barrier and enter the brain parenchyma. Our result showed that, SRL-modified nanoparticles provide a safe and effective nanocarrier for brain gene delivery. Ó 2015 Published by Elsevier B.V.

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Keywords: Blood–brain barrier SRL peptide Gene delivery PAMAM dendrimer 70

1. Introduction ⇑ Corresponding author at: Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, PO Box 19395-5719, Tehran 1985717443, Iran. E-mail addresses: [email protected], [email protected] (B. Kazemi).

The main obstacle in drug delivery, including deliver of the genetic material (e.g. DNA) is permeation through blood–brain barrier (BBB). This problem has made the treatment of nervous system

http://dx.doi.org/10.1016/j.ejps.2015.06.024 0928-0987/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: Zarebkohan, A., et al. Synthesis and characterization of a PAMAM dendrimer nanocarrier functionalized by SRL peptide for targeted gene delivery to the brain. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.06.024

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diseases such as brain disease rather difficult. Blood–brain barrier inhibits most of drugs and genetic materials (almost 98%) from reaching the brain and allows passage only to particles that are either small (less than 400 Da) or are very lipophilic; hence failure in treatment of most nervous system diseases (Pardridge, 2010; Pang et al., 2011). Nowadays, nanotechnology has provided solutions to such problems and different nanoparticles and supramolecular nanodevices have been developed to improve permeation of drugs and even genetic materials through different cellular and epithelial barriers in a non-invasive manner (Paolino et al., 2011; Celia et al., 2011). One class of these nanoparticles, which have been considered by researchers as a great tool for gene delivery, are PAMAM dendrimers (Li et al., 2011; Xin et al., 2011). As PAMAM dendrimers have primary surface amines, such nanoparticles have a good potential and capacity for attaching to different types of negatively charged drugs, including gene segments, plasmids and several types of other ligands (Li et al., 2011). However, these carriers cannot permeate BBB easily. One of the methods that has been used to solve this problem is attachment of certain ligands to the surface of these nanoparticles to facilitate their passage through BBB. These ligands can transport the nanocarriers by identifying the surface receptors of brain capillary endothelial cells and also through transcytosis (Huang et al., 2007). There are several strategies for mass transport across the BBB including: passive diffusion, carrier mediated transport (active and facilitated) and endocytosis/transcytosis (Zlokovic, 2008). Passive diffusion depends on lipophilicity, and diffusivity of the molecule and is driven by concentration gradient across BBB, hence does not require energy (Cohen and Bangham, 1972; Camenisch et al., 1998). Carrier mediated transport is also a kind molecular transport mediated by carriers (transporters) in the membrane (Crone, 1965). The most attractive route for nanotherapeutics delivery to brain is endocytosis/transcytosis pathways that although occurs naturally, it can be facilitated by using ligands that attach the nanoparticle to the membrane surface or can induce internalization. For this purpose several types of ligands have been developed. Antibodies (Abs) against the highly expressed receptors at the BBB such as insulin and transferrin are one of them (De Boer et al., 2003; Zhang et al., 2002; Mamot et al., 2005). Monoclonal Abs have high specificity to the receptor targets, but there high molecular weight and complicated structure have limited their application. Cell-penetrating peptides are another ligands attached to the nanoparticles to promote drug trafficking across BBB (Schwarze et al., 1999). Some of these peptides can trigger the severe immunogenic responses. Endogenous derived peptides such as apolipoprotein A (ApoA) and apolipoprotein E (ApoE) Goppert and Muller, 2005; Kreuter et al., 2003 and other substrates including thiamine, folate, glycosides and lactoferrin (Lockman et al., 2003; Eavarone et al., 2000; Mora et al., 2002) were evaluated for targeting the BBB. These molecules are naturally found in human and therefor they do not evoke immunological response. However, receptor specificity of these molecules is less than other types of ligands. To increase their specificity and optimize other properties, synthetic peptides have been used as a new class of ligands for targeting the nanoparticles to BBB (Demeule et al., 2008). In most cases, the mechanism underlying transport of nanoparticles decorated by these peptides is endocytosis/transcytosis pathway. Advantages of these peptides include relatively low cytotoxicity and immunogenicity, and biodegradable properties. Other advantages of peptides over other ligands include well-established chemistry, simplicity of synthesis and characterization (Gaumet et al., 2008; Gynther et al., 2009). Other techniques have also been used to improve CNS drug delivery including efflux transporter inhibitors (for increase brain accumulation of drugs by inhibition of drug efflux to out of cite)

(Salama et al., 2005), positively charged nanocarriers (due to better interaction with cell membranes) (Kumagai et al., 1987; Lu et al., 2007) and transcellular membrane permeability pathways (by using pharmacological agents or a hyperosmotic approach). Application of drug through the nasal cavity (Mistry et al., 2009) and transient disruption of BBB by focused ultrasound and gas microbubbles are other strategies and have been applied for genes and proteins delivery through BBB (Hynynen et al., 2005). Generally, the three main routes through which molecules can be transported to the brain are carrier-mediated transport (CMT), receptor-mediated transcytosis (RMT) and adsorptive-mediated transcytosis (AMT). In case of CMT, the transport is through luminal and abluminal brain capillary membranes so that the nanocarrier are identified as an endogenous ligand and is not rejected (Duffy et al., 1988). RMT is dependent on selective absorption by special attachment to the receptors or small peptides in order to be transferred through luminal membrane of BBB endothelial cells. Drug targeting by nanoparticles using TAT peptide (Li et al., 2011; Lo and Wang, 2008) or CTP (cytoplasmic transduction peptide) (Sauer et al., 2005); have used RMT mechanism. Also peptide targeted quantum dot have been used in cancer cells imaging (Xu et al., 2008). AMT works through electrostatic interactions between positively charged penetrants and negatively charged surface of the cell membrane. This system is dependent on a high level of positive charges on the nanoparticles; therefore, dendrimers are a good candidate for this purpose (Sauer et al., 2005). Several nanoparticles are delivered through BBB using RMT and AMT pathways using peptides as the targeting moiety including angiopep-2 (Shao et al., 2010; Ke et al., 2009), Tet1 (Kwon et al., 2010), TGN (Li et al., 2011), lactoferrin (Hu et al., 2009) and transferrin (Gan and Feng, 2010). In Pasqualini and Ruoslahti (1996) designed a 9 amino acid cyclic peptide with CLSSRLDAC sequence and showed its efficiency in penetrating the brain capillary endothelial cells (BCECs). In the present investigation, we used two linear forms of this peptide in two sequences of CLSSRLDA and LSSRLDAC. The differences between the peptide used here and that reported by Pasqualini include: (A) the present peptide is linear while the original peptide is cyclic. This modification was made to facilitate the attachment of the peptide to the nanoparticle. (B) The peptides, that are called SRL peptide here, were then used here to target DNA-loaded PAMAM dendrimers to the brain. To the best of our knowledge, these peptides have not been used as a targeting ligand in gene-delivery or PAMAM delivery by other investigators.

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2. Materials and methods

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Plasmid pEGFP-N1 (Clontech, Palo Alto, CA, USA) was purified using QIAGEN Plasmid Mega Kit (Qiagen GmbH, Hildden, Germany). Peptide SRL with two sequences (CLSSRLDA and LSSRLDAC) was purchased from Biomatic Company (Wilmington, USA) with a purity percentage of 99%. PAMAM G4 dendrimer was synthesized by in situ branch cell method, which is a two-step iterative process for constructing poly(amidoamine) (PAMAM) dendrimers possessing either terminal ester or amine groups. This method involves (a) alkylation with methyl acrylate, and (b) amidation with ethylenediamine (Esfand and Tomalia, 2002). Lactoferrin, filipin complex (from Streptomyces filipinesis),

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L-polylysin

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(30,000–70,000 MW) and phenylarsine oxide, were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bifunctional PEG (NHS-PEG-MAL, MW 1000) was purchased from Nanocsinc (Boston, USA). BODIPY fluorophore (4,4-difluoro-5,7-dime thyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, sulfosuccinimidylester, sodium salt) was purchased from Molecular Probes (Eugene, OR, USA).

Please cite this article in press as: Zarebkohan, A., et al. Synthesis and characterization of a PAMAM dendrimer nanocarrier functionalized by SRL peptide for targeted gene delivery to the brain. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.06.024

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2.1. Animals

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Balb/c male mice that were 4–5 weeks old and weighed 20–25 g were bought from the Department of Laboratory Animals of the Razi Vaccine and Serum Research Institute in Iran and were kept in standard circumstances. All the animal researches were carried out according to the assessment guide of the ethic committee of Shahid Beheshti University of Medical Sciences.

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2.2. Cell line

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Brain capillary endothelial cells of rats (BCEC) were received as a gift from Dr. Aghaei (Neuroscience Research Center, Shahid Beheshti University of Medical Sciences) and were cultured based upon the guidelines of the ATCC. BCECs were expanded and cultured in a Rat Brain Endothelial Cell Growth Medium (Cell Applications Inc., San Diego, CA, USA) containing 20% fetal calf serum (FCS), 100 mg/ml epidermal growth factor, 2 mmol/l

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L-glutamine,

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20 mg/ml heparin, 40 mU/ml insulin, 100 U/ml penicillin and 100 mg/ml streptomycin at a temperature of 37 °C and in an atmosphere containing 5% CO2. 2.3. Synthesis of PAMAM derivatives and characterization of nanoparticles 2.3.1. Conjugation of bifunctional PEG to dendrimers PAMAM derivatives were created according to the methods mentioned in the previous articles (Ke et al., 2009; Pardridge, 2005). PAMAMs, with molar rations of 1:2, 1:4 and 1:6 in PBS (pH 8.0), reacted to NHS-PEG1000-MAL for 2 h in room temperature. In this stage, the PAMAM primary amine groups react exclusively to terminal NHS groups of bifunctional PEG. In order to separate the unreacted PEGs, (5 and 10 kDa) (Amicon) filters were used. 2.3.2. PAMAM–PEG–SRL synthesis The resulted conjugates redissolved into PBS (pH 7.0). Then, PAMAM–PEG with molar ratio of 1:1, 1:2 and 1:3 (mol/mol) in PBS (pH 7.0), reacted with SRL peptide at room temperature for 12 h. Terminal MAL groups of PAMAM–PEG reacted exclusively to SRL thiol groups (Ke et al., 2009). 2.3.3. BODIPY-labeled nanoparticles synthesis In order to synthesize labeled conjugates with BODIPY, PAMAM was initially reacted by BODIPY in 100 mM NaHCO3 for 12 h in 4 °C and using the above method purified. Other conjugates (BODIPY-labeled PAMAM, and BODIPY-labeled PAMAM–PEG–SRL) were also made using the method mentioned above (Ke et al., 2009). 2.3.4. Characterization of synthesized nanoparticles NMR and Zeta sizer were used in order to study the exact chemical and physical characteristics of the synthesized nanoparticles. In order to analyze NMR, PAMAM–PEG–MAL and PAMAM–PEG– SRL solutions were initially turned into powder; they were then dissolved in D2O and analyzed in 400 MHz spectrometer. Size of the particle and the Zeta potential of nanoparticles were studied using dynamic light scattering and zeta plus analyzer (Zeta-sizer, Malvern nanozs, U.K.). As application of AFM in such investigations is usual and has been employed by other investigators (Shao et al., 2010; Navarro and de ILarduya, 2009; Luo et al., 2012; Beloor et al., 2012) we had concluded that this technique is reliable enough. Atomic Force Microscope (Digital Instrument, Santa Barbara, CA) was used for imaging the nanoparticles and nanoparticle–gene complex. AFM was used in tapping mode (Shao et al., 2010).

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Schematic presentation of PAMAM–PEG and PAMAM–PEG–SRL synthesis is shown in Fig. 1.

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2.4. Preparation of dendrimer/DNA nanoparticles

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PAMAM and PAMAM–PEG–SRL were freshly synthesized and diluted in distilled water. Then, DNA solution (100 mg DNA/ml) in sodium sulfate solution (50 mM) was added to nanoparticles solution with specific weight ratios (1:10, 1:5, 5:1, 10:1, PAMAM/DNA w/w) and was vortexed slowly for 30 min (37 °C). After that, the complex was incubated in room temperature for 30 min so that self-assembly would be possible. Agarose gel electrophoresis was carried out in order to prove the formation of nanoparticle–DNA complex and the stability of the complex (Ke et al., 2009).

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2.5. Electrophoresis of dendrimers/DNA NPs

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4 different ratios of PAMAM–PEG–SRL/DNA complex (1:10, 1:5, 5:1, 10:1, PAMAM/DNA w/w) were synthesized and electrophoresis in 0.7% agarose gel done. Electrophoresis was carried out for 30 min under 100 V/cm and the results were recorded using gel doc. It was done to proving formation and stability of nanoparticle/DNA the complex (Kuang et al., 2013).

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2.6. Preparation of endocytosis inhibitors

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Filipin complex and phenylarsine oxide were dissolved in dimethyl sulfoxide (DMSO) and were then diluted in PBS (pH 7.4) up to (0.5 mg/ml). SRL (100 mg/ml), L-polylysin (25 mg/ml), colchicines (2.5 mM) or lactoferrin (1 mg/ml) were prepared in water and were then diluted in PBS (pH 7.4) Ke et al., 2009.

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2.7. Cellular uptake of dendrimers

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First, BCECs were cultured for 48 h with a density of 2  104 cells/well in 24-well plates (Corning-Coaster, Sigma, USA), and their confluency and morphology were assessed. After that, BCECs were incubated with 1 lM of BODIPY-labeled PAMAM or 0.2, 0.5 and 1.0 lM of BODIPY-labeled PAMAM– PEG–SRL for 30 min in 37 °C with a final concentration of PAMAM 1 lM. Also, in order to study the energy dependency of the nanoparticles entrance to target cells, BODIPY-labeled PAMAM–PEG–SRL was exposed to BCECs with 1 lM density in 4 °C. Then, the solution was removed and cells was washed with cold PBS (pH 7.4) three times and observed using fluorescent microscope (Olympus, Osaka, Japan). For quantitative analysis of cellular uptake of PAMAM–PEG–SRL in different concentration, BCECs with a density of 8  104 cells/well in 6-well plates (Corning-Coaster, Sigma, USA) were cultured for 72 h and were then studied under microscope for their confluency and morphology. Then, BCECs were incubated with 0.2, 0.5 and 1 lM from BODIPY-labeled PAMAM–PEG–SRL for 30 min. Similar to the previous stage, the cells were washed three times with PBS (pH 7.4); then, in order to obtain the cellular plaque, the treated cells were trypsinized and centrifuged for 8 min in 1600 rpm. The acquired plaque was resuspended in PBS (pH 7.4) again and was analyzed by flow cytometer (FACS Calibur, BD Biosciences, Bedford, MA, USA) equipped with Argon Ion Laser (488 nm) as a stimulating source of fluorescent. For each sample, 10,000 event were collected and analyzed using WinMDI software. The cultured BCECs under normal conditions were considered as controls. The live cells were considered as gates for most of the cells and only the cells situated in this gate were analyzed. The average density of the fluorescent of the cells was calculated as histogram plot (Ke et al., 2009).

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Fig. 1. Presentation of PAMAM–PEG and PAMAM–PEG–SRL synthesis.

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2.8. Cellular uptake mechanism of PAMAM–PEG–SRL

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Initially, BCECs were seed at a density of 2  104 cells/well in 24-well plates and were cultured using the above mentioned method. After controlling the morphology and the confluency of the cells, the filipin complex (0.5 mg/ml), phenylarsine oxide (2.5 mM), L-polylysin (25 mg/ml), colchicines (2.5 mM) or lactoferrin (1 mg/ml) were added to each well and were incubated for 10 min. Then, the compounds were removed and BODIPY-labeled PAMAM–PEG–SRL nanoparticles were added to the cells with the above mentioned concentration. After 30 min, the solution was removed and the cells were washed three times with cold PBS (pH 7.4) and were observed under fluorescent microscope (Ke et al., 2009).

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2.9. Efficiency of gene transfer into BCECs

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Plasmid DNA was labeled covalently using fluorescent color ethidiummonoazide bromide (EMA). Briefly, the new plasmid DNA solution (1 mg/ml in TE buffer, pH 7.0) was diluted with the aqueous solution EMA until reaching the density of 0.1 mg/ml and was incubated for 30 min in darkness. Then, the solution was deposited to the final density of 30% (v/v) adding ethanol. The sediment was collected by a centrifuge at 12,000 rpm for 20 min and was redissolved in sodium sulfate 50 mM. EMA-DNA solution was used to prepare nanoparticles through the above

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method. BCECs were seed at a density of 2.0  104 cells/well in 24-well plates and were incubated for 48 h. After that, the nanoparticles (PAMAM or PAMAM–PEG–SRL) containing EMADNA were added to the cells (Ke et al., 2009). 30 min later, the solution was removed, cells were washed three times with PBS (pH 7.4) and was then observed under fluorescent microscope.

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2.10. Cellular uptake mechanism of dendrimer/DNA NPs

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BCECs were seed at a density of 2  104 cells/well in 24-well plates and were cultured using the above method. After controlling morphology and confluency of the complex filipin (0.5 mg/ml), phenylarsine oxide (2.5 mM), L-polylysin (25 mg/ml), colchicines (2.5 mM) or lactoferrin (1 mg/ml) were added to each well and were incubated for 10 min (Ke et al., 2009). Then, the mixtures were removed and the nanoparticles PAMAM–PEG–SRL containing EMA-DNA were added with the above mixtures (quantitatively equal). After 30 min, the surface solution of the cells was removed and the cells were washed three times with cold PBS (pH 7.4) and were observed under fluorescent microscope.

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2.11. In vitro cytotoxicity assay

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The cellular toxicity of the nanoparticles complex was studied using MTT assay. In brief, the BCECs with 10,000 cells/well density were cultured in 96-well plates. PAMAM, PAMAM–PEG and

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PAMAM–PEG–SRL nanoparticles with different concentrations (10, 50, 100, 150, 200 lg) containing 1 lg DNA were added to the cells in a serum free medium and were incubated for 4 h. Then, supernatant was removed and 20 ll MTT solution (5 mg/ml) was added to each well. Untreated cells as a negative control was used for comparison. In the next stage, the medium was precisely removed and 200 ll (DMSO) was added to each well. Then, the plate was incubated for 15 min in 37 °C and formazin absorption in 570 nm was measured using micro plate reader. This experiment was repeated 3 times and the relative cell viability was shown in the form of the percentage of live cells to the untreated control cells. For Assay, 48 h of experiments similar to the above were carried out; the only difference was that 100 ll of the culture medium of BCEC was added to each well after 4 h (Kuang et al., 2013).

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The intracellular distribution of nanoparticles was studied using confocal microscopy. BCECs with the density of 104 cells/well were seeded in 35 mm glass-bottom culture dish and were cultured in 37 °C for 48 h in the presence of 5% CO2 and were then cultured in a serum free media for 2, 30 and 60 min with the PAMAM– PEG–SRL/DNA complex (5 lg DNA/well, N/P = 10). After the mentioned time, the cells were washed with cold PBS and were incubated with 50 nM LysoTracker Green (Molecular probes Inc.) for 30 min and with DAPI for 10 min. Then, the cells were washed three times with cold PBS and were fixed with paraformaldehyde and were observed under laser scanning confocal laser microscopy (Qian et al., 2013).

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The nanoparticles containing pEGFP (10:1, PAMAM to DNA, w/w) with a dose of 50 lg DNA/mice were injected into the tail veins of the mice. 2 h later, the mice were imaged using Cri in vivo imaging system (Ke et al., 2009).

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PAMAM–PEG–SRL and PAMAM nanoparticles containing pEGFP (10:1, PAMAM to DNA, w/w) with 50 lg DNA/mouse dose, were injected into the tail veins of the mice. After 48 h, the animals sacrificed under the influence of diethyl ether and they were decapitated. Brains were removed from the skulls and were fixed in Paraformaldehyde 4% for 48 h; they were then subsided in 15% sucrose PBS for 24 h and in 30% sucrose for 48 h. Then, the brains were frozen in OCT embedding atmosphere in 80 °C. The frozen sections with 20 lm thickness were prepared using cryotome Cryostat (Leica, CM 1900, Wetzlar, Germany) and were stained for 10 min at room temperature using DAPI 300 nM. The sections were then washed with PBS pH 7.4 two times and were observed using fluorescent microscope (Ke et al., 2009).

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All the quantitative assessments were collected using quadruplicate method and the experiments were repeated 3 times. The data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA, using statistical software.

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3. Results

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3.1. Characterization of PAMAM–PEG–SRL

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In NMR spectrum (Fig. 2), the solvent peak D2O was observed in 4.78 ppm. Methylene protons of PAMAM branch units contained several peaks between 2.0 and 3.2 ppm. The repetitive units of PEG showed a sharp peak in 3.7 ppm. The specifying peak of the MAL group was also observed in PAMAM–PEG in 6.13 ppm (Fig. 2B). However, MAL peak was not observed in the spectrum related to PAMAM–PEG–SRL which indicates the attachment of MAL group to SRL thiol group, however, the repetitive units of PEG kept showing the sharp peak of 3.7 ppm situation (Fig. 2B). The percentage of PEG attachment to PAMAM was calculated from the integral ratio of –NHCH2CH2–(C/c, 2.0–3.2 ppm) to PEG (i, 3.7 ppm), by the following formula, PAMAM–PEG = 2(C/c)/3(i)) Zeng et al., 2011. Our results shown that the attachment efficiency was 80% (about 51.2 PEG molecule per each dendrimer). The attachment of BODIPY, PEG and SRL to the dendrimer was confirmed by UV spectroscopy and the FTIR techniques (Supplementary data). DLS (Table 1) and AFM (Fig. 3A) were carried out in order to study the size and the morphology of nanoparticles and their complex with the genes. Results showed that the mean size of G4 PAMAM has increased from about 4 nm to 32 nm in PAMAM– PEG–SRL (Fig. 3A) which is due to the attachment of SRL peptide to PAMAM–PEG. But, the zeta potential of G4 PAMAM dendrimer has not really change due to attachment with peptide SRL (due to the neutral charge of the peptide). Also, agarose gel electrophoresis was proved complex formation of nanoparticles/DNA and its stability (Fig. 3B).

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The BODIPY-labeled nanoparticles were used in cellular uptake study and the fluorescent images were analyzed. In order to investigate the concentration dependency of nanoparticles entrance into the BCEC cells, different concentrations of (0.2–1 lM) BODIPY-labeled PAMAM–PEG–SRL were used over 30 min cellular exposure. As can be seen in Fig. 4, at concentration of 1 lM, an outstandingly higher cellular uptake was observed for PAMAM–PEG– SRL compared to PAMAM (Fig. 4A). As the concentration of PAMAM increased from 0.2 to 1 lM, the percentage of positive BODIPY cells increased from 48.8% to 99.7% and the average intensity of the fluorescent increased from 39.3 to 66 (Fig. 4B). Also, the nanoparticles cellular uptake increased by increase of peptide on the nanoparticles (Supplementary data) which could be due to recognition of the nanoparticles by more receptors. Our data showed that, using the inhibitors such as filipin,

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or lactoferrin (Fig. 5B–E) caused the inhibition of cellular uptake for PAMAM–PEG–SRL. These inhibitors were used in order to study the mechanism inhibiting the entry of nanoparticles into the cells. Among these inhibitors, lactoferrin has the highest level of inhibition (Fig. 5B). As a ligand, lactoferrin is known as having a high affinity to LRP (Fillebeen et al., 1999; Tamaddon et al., 2007). The cellular uptake of BODIY-labeled PAMAM–PEG–SRL at 4 °C was much less than 37 °C that could be considered as another reason for energy-dependency of nanoparticles cellular uptake; endocytosis (Fig. 5F) Tamaddon et al., 2007. Results also indicated that the cellular uptake of modified nanoparticles is dose dependent. Also, no difference was observed between the cellular absorption of the modified nanoparticles and the two forms of SRL peptide (N or C terminal added cysteine residue) (the data are not shown due to the similarity of fluorescent images).

Please cite this article in press as: Zarebkohan, A., et al. Synthesis and characterization of a PAMAM dendrimer nanocarrier functionalized by SRL peptide for targeted gene delivery to the brain. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.06.024

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Fig. 2. NMR spectrum of PAMAM–PEG (A) and PAMAM–PEG–SRL (B). The percentage of PEG attachment to PAMAM was calculated from the integral ratio of –NHCH2CH2–(C/ c, 2.0–3.2 ppm) to PEG (i, 3.7 ppm), by the following formula, PAMAM–PEG = 2(C/c)/3(i)). MAL: Maleimide.

Table 1 Particle size and zeta potential of G4 and PAMAM–PEG–SRL with or without BODIPY (n = 3). Nanoparticle

Mean size (mean ± SD, nm)

Zeta potential (mV)

PDI

G4 G4-PEG G4-PEG-SRL G4-PEG-SRL/DNA (N/P = 10:1)

3.8 ± 0.6 6 ± 0.5 32 ± 7 150 ± 62

3.8 ± 0.8 2 ± 0.4 2.2 ± 0.3 20 ± 4

0.55 0.32 0.33 0.62

477

3.3. Cellular uptake of DNA-containing particles, mechanistic study

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Results showed that amongst the three PAMAM:PEG:SRL compositions used here (1:2:1, 1:4:2 and 1:6:3 M ratios) the 1:6:3 ratio showed the highest level of uptake (Supplementary data). Also, it was observed that cellular uptake of PAMAM–PEG–SRL/DNA (Fig. 4) is more than PAMAM/DNA. The findings showed that all

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the inhibitors prevent the entry of nanoparticles into the cells. As can be seen in Fig. 6, lactoferrin, as an LRP inhibitor, inhibits the delivery and shows the highest level of inhibition (Fig. 6F).

483

3.4. Cellular localization of PAMAM–PEG–SRL/DNA NPs

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Confocal microscopy was used in order to study the intracellular distribution of PAMAM–PEG–SRL/DNA nanoparticles. Endocytosis is a process where molecules are internalized as vesicles into the cytoplasm, called endosomes (Simionescu et al., 2002). Drug should escape endosome to be able to work in the cell; otherwise, they will end in lysosomes, where they will be destroyed. Therefore escape in early stages (endosomal escape) is necessary for drugs that are entered the cells via endocytosis. The cellular uptake of EMA-labeled PAMAM–PEG–SRL/DNA complexes occurred in a time-dependent manner. In Fig. 7, the small red dots represent DNA, whereas the small green dots represent LysoTracker green.

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Fig. 3. (A) Topographic AFM images of PAMAM–PEG–SRL, area of scanning 0.4  0.4 lm and (B) PAMAM–PEG–SRL/DNA nanoparticles, area of scanning 2  2 lm. (C) Agarose gel electrophoresis of complexes at various weight ratios. Con: naked plasmid, Den: Dendrimer.

Please cite this article in press as: Zarebkohan, A., et al. Synthesis and characterization of a PAMAM dendrimer nanocarrier functionalized by SRL peptide for targeted gene delivery to the brain. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.06.024

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Fig. 4. (A) Cellular internalization of BODIPY-labeled PAMAM–PEG–SRL in the concentration of 0.2 lM (G–I), 0.5 lM (J–L), 1.0 lM (M–O) and 1.0 lM PAMAM-BODIPY (D–F) was examined after 30 min incubation. Normal BCECs served as the control (A–C). Green: BODIPY, blue: DAPI. Original magnification: 200. (B) Flow cytometry analysis of BCECs after a 30 min incubation. Histogram of normal BCECs (A), PAMAM-BODIPY 1 lM (B), different concentration of PAMAM–PEG–SRL 0.2 (C), 0.5 (D) and 1 lM (E). (C) After setting a gate according to the control the number of BODIPY-positive cells were analyzed. % BODIPY-positive cells (up) and mean fluorescence intensity (down) was shown, respectively. P value

Synthesis and characterization of a PAMAM dendrimer nanocarrier functionalized by SRL peptide for targeted gene delivery to the brain.

Blood-brain barrier inhibits most of drugs and genetic materials from reaching the brain. So, developing high efficiency carriers for gene and drug de...
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