Aptamer-targeted delivery of Bcl-xL shRNA using alkyl modified PAMAM dendrimers into lung cancer cells

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International Journal of Biochemistry and Cell Biology

journal homepage:www.elsevier.com/locate/biocel

Research paper

Aptamer-targeted delivery of Bcl-xL shRNA using alkyl modi fi ed PAMAM dendrimers into lung cancer cells

Sara Ayatollahi

^a

, Zahra Salmasi

^b

, Maryam Hashemi

^b

, Saeedeh Askarian

^c,d

, Reza Kazemi Oskuee

^e

, Khalil Abnous

^a,⁎

, Mohammad Ramezani

^a,f,⁎

aPharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

bNanotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

cResearch Center of Advanced Technologies in Medicine, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran

dDepartment of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

eTargeted drug delivery research center, Mashhad University of Medical Sciences, Mashhad, Iran

fDepartment of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

A R T I C L E I N F O

Keywords:

Targeted delivery Aptamer Bcl-xL shRNA Non-viral vector PAMAM

A B S T R A C T

RNAi-based gene therapy has been recently considered as a promising approach against cancer. Targeted de- livery of drug, gene or therapeutic RNAi-based systems to tumor cells is one of the important issues in order to reduce side effects on normal cells. Several strategies have been developed to improve the safety and selectivity of cancer treatments including antibodies, peptides and recently aptamers with various attractive characteristics including higher target specificity, affinity and reduced toxicity. Here we described a novel targeted delivery platform comprising modified PAMAM with 10-bromodecanoic acid (10C) and 10C-PEG for improvement of transfection efficiency, AS1411 aptamer for targeting nucleolin ligand on target cancer cells and shRNA plasmid for specific knockdown of Bcl-xL protein. Modified vector could significantly improve the transfection efficiency even after covalent or non-covalent aptamer binding compared to the non-targeted vector in A549 cells. The results of gene silencing and apoptosis assay indicated that our targeted shRNA delivery system could efficiently down-regulate the Bcl-xL expression up to 25% and induce 14% late apoptosis in target cancer cells with strong cell selectivity. This study proposed a novel targeted non-viral system for shRNA-mediated gene-silencing in cancer cells.

1. Introduction

RNA interference (RNAi) is a process with high specificity for silencing or knockdown of gene expression by inactivation of its related mRNA through double strand RNA. RNAi effect can be meditated through two methods: synthetic small interfering RNA (siRNA) or plasmid-based short hairpin RNA (shRNA). shRNA has designed to produce continuously double-strand RNA in the nucleus of transfected cells for which low copy numbers of shRNA can provide more efficient and continual effects with less off-target gene silencing (Moore et al., 2010; Rao et al., 2009).

However, like other plasmids, shRNA could not efficiently pass cellular membrane and get introduced into the nucleus. Therefore, one of various efficient nano-carriers is required for shRNA delivery into target cells (Askarian et al., 2015; Mokhtarzadeh et al., 2017). Among different types of nano-carriers, dendrimers have high potential in gene/drug delivery due to their well-defined, mono-disperse structure as well as cationic surface charge and surface functionality (Abbasi et al., 2014). With these

great features, dendrimers could efficiently interact and condense nucleic acids as well as undergo different surface modifications for targeted de- livery. Different dendritic structures such as polyamidoamine (PAMAM), poly (propylene imine) (PPI), poly-L-lysine, poly (etherhydroxylamine) (PEHAM), poly (esteramine) (PEA) and polyglycerol have been used as drug/gene delivery systems (Madaan et al., 2014). Among various den- drimers, PAMAM has been widely considered as an efficient carrier for gene/drug transfer and recently for RNAi delivery (Kesharwani et al., 2015; Madaan et al., 2014; Wu et al., 2013).

In this study, a novel targeted delivery system has been designed consisting of modified PAMAM with either 10-bromodecanoic acid (10C) or 10 C-PEG, AS1411 aptamer for targeting nucleolin ligands and shRNA plasmid for specific knockdown of Bcl-xL protein.

The Bcl-2 family members could be classified into pro-apoptotic (BAX, BAK and BOK), anti-apoptotic (Bcl-2, Bcl-xL and MCL1) and BH3-only proteins. Cellular fate is determined by considering the balance between these groups. Protecting the outer mitochondrial membrane and inhibiting

http://dx.doi.org/10.1016/j.biocel.2017.10.005

Received 24 July 2017; Received in revised form 7 October 2017; Accepted 10 October 2017

⁎Corresponding authors at: Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

E-mail addresses:abnouskh@mums.ac.ir(K. Abnous),ramezanim@mums.ac.ir,ramazanim@gmail.com(M. Ramezani).

Available online 12 October 2017

1357-2725/ © 2017 Elsevier Ltd. All rights reserved.

MARK

release of cytochrome c is the main role of anti-apoptotic proteins. High Bcl- xL expression has been reported in different solid tumors such as neuronal tumors, adenocarcinoma, bladder cancer, and gastric cancer (Kang and Reynolds, 2009; Kirsh et al., 1998; Krajewski et al., 1997). Therefore, se- lective silencing of Bcl-xL could induce apoptosis in cancer cells.

Targeted delivery of desired genes/drugs to tumor environment has recently emerged as effective strategy to improve specificity of cancer treatments as well as to decrease toxicity on normal cells (Akhtar et al., 2014; Catuogno et al., 2016; Kurosaki et al., 2012; Yu et al., 2010). In different studies, various targeting ligands have been used including antibodies, peptides and small molecules (Askarian et al., 2017;

Friedman et al., 2013; Yao et al., 2016). Aptamers as specific targeting agents are short and non-coding single stranded oligonucleotides (DNA or RNA) which obtained from a technology called systematic evolution of ligand exponential enrichment (SELEX) (Chen et al., 2015; Ellington and Szostak, 1990; Tuerk and Gold, 1990). Compared to antibodies, aptamers possess several advantages, including small size that allows for rapid penetration into the tumor cells, low immunogenicity, easy synthesis and modification, considerable stability over wide range of pH (4–9), temperature and organic solvent without loss of activity and low cost (Sultana et al., 2013; Sun et al., 2014; Wang et al., 2015).

Aptamer-functionalized systems could be applied for biosensing, tar- geted drug or gene delivery purposes and cancer cell imaging. Chen et al. have developed a new strategy for selection of RNA aptamer which specifically targeted one of the important p53 mutations (p53R175H). In vitro and in vivo studies showed that this aptamer could induce apoptosis and reduce the growth rate and the migration capability of human lung cancer cells harboring p53R175H (Chen et al., 2015). In other studies, various aptamer-conjugates based on den- drimers have been designed and used as gene delivery systems to target specific cancer cells (Wang et al., 2015; Wu et al., 2014).

AS-1411 is a 26-mer DNA aptamer which binds specifically to nu- cleolin, a protein over-expressed on various cancer cells including acute myelogenous leukemia (AML) (Mongelard and Bouvet, 2010), MKN45, KATOIII, AGS (Watanabe et al., 2010) and A549 (Ireson and Kelland, 2006). It was reported that nucleolin has been detected only in the nucleus of normal cells whereas it was found either in the nucleus or cytoplasm of malignant cells. As a result, AS1411 aptamerfirst specifically binds to nucleolin over-expressed on the surface of cancer cells and then inter- nalized (Ireson and Kelland, 2006; Soundararajan et al., 2008).

Considering the overexpression of nucleolin in various cancers, in this study, AS-1411 aptamer was used as targeting agent.

With these perspectives, we designed and developed targeted shRNA delivery system based on alkyl modified PAMAM dendrimer conjugated to AS1411 aptamer for the selective knockdown of Bcl-xL expression in nucleolin positive cancer cells.

2. Materials and methods 2.1. Materials

PAMAM dendrimer solution of generation 4 in methanol and 10- bromodecanoic acid were obtained from Sigma-Aldrich (Schnelldor, Germany). Hetero-functional poly(ethylene glycol) (NH2–PEG–COOH) (MW∼3400) was purchased from NanoCS (New York). AS1411 ap- tamer was synthesized by MicroSynth (Switzerland) and PAGE purified.

The sequence is 5ʹTTGGTGGTGGTGGTTGTGGTGGTGGTGG3ʹ(5′-NH2).

pGeneClip™hMGFP-Bcl-xL shRNA (sequence = CTCACTCTTCAGTC GGAAATGACCAGACA) (size:5.2 Kb) was purchased from Qiagen (Qiagen, Hilden, Germany). A549 and L929 cell lines were obtained from Pasteur Institute of Iran. Cells were cultivated in RPMI 1640 (Gibco), supplemented with 100 units/ml penicillin–streptomycin (Sigma) and 10% fetal bovine serum (Gibco, USA).

2.2. Conjugation of 10-bromodecanoic acid (10C) and10C-PEG chains to PAMAM

The synthesis of the PAMAM derivatives was performed according to the method described in our previous studies (Ayatollahi et al., 2015; Hashemi et al., 2015). Briefly, 10-bromodecanoic acid chain was conjugated to COOH- PEG-NH2 by amid bond formation. Then, either 10C or 10C-PEG and 10Cchains in chloroform were added dropwise to the stirred chloroform so- lution of PAMAM dendrimer to substitute 3 and 30% of primary amines, respectively. After 24 h, the solvent was evaporated under reduced pressure;

the residue was dissolved in water and dialyzed against distilled water (8–12 kDa cut offdialysis membrane) for three days and then lyophilized.

2.3. Covalent and non-covalent binding of aptamer to modified PAMAM For covalent conjugation of aptamer, PAMAM derivative was dis- solved in DNase–RNase free water (Sigma) with equal molar ratio of EDC/NHS and stirred for 30 min at room temperature. After activation of carboxyl groups of vector, 5′NH2-modified anti AS1411 aptamer was added (5% weight of vectors) and left for 2 h at room temperature with gentle stirring. Finally, the product was washed three times with DNase/RNase-free water by Amicon filters (Millipore) and used im- mediately in the next steps.

For non-covalent (physical) conjugation of aptamer, DNA aptamer (5% weight of vector) was added to selected vector solution in RNase/

DNase free water and stirred for 2 h at room temperature.

2.4. Confirmation of vector-aptamer conjugation

To investigate vector-aptamer conjugation, agarose gel electro- phoresis even in the presence or absence of heparin was used. Briefly, 40μl of each sample was incubated with 3μg heparin for 5 min. Then, 5μl loading dye was added and samples were run on 1% agarose gel containing 0.03% EtBr. Electrophoresis was performed at 80 V in run- ning buffer (Tris-acetate-EDTA) for 30 min. The results were analyzed by Alliance 4.7 gel doc (Uvitec, UK).

2.5. Particle size and zeta potential measurements of aptamer-polyplex conjugate

The particle size and zeta potential of the polyplex composed of vector/pDNA/aptamer conjugate prepared in HBG buffer (HEPES-buf- fered glucose containing 20 mM HEPES, 5% glucose, pH 7.4) was measured using dynamic light scattering (DLS) and laser Doppler ve- locimetry (LDV), respectively by a Malvern Nano ZS instrument and DTS software (Malvern Instruments, UK). Modified PAMAM before and after aptamer conjugation was added to DNA plasmid (5μg) in HBG.

The mixture was incubated for 30 min at room temperature. The results were reported as mean ± SD of 3 runs.

2.6. Evaluation of target receptor expression on the surface cells

Presence of desired receptor on the surface cells (A549) was in- vestigated by immunocytochemistry assay. Briefly, one drop of cell sus- pension was placed on each slide and centrifuged at 2500 rpm for 5 min to form monolayer separated cells. Afterfixation, cells were incubated with primary antibody against nucleolin (Abcam) followed by peroxidase-la- beled anti-rabbit IgG (Goat anti-Rabbit IgG H & L, Abcam) and the pre- sence of desired receptors was investigated using light microscope.

2.7. Cell culture

Human lung adenocarcinoma cell line, A549 (ATCC CCL-185) and fibroblast cell line, L929 (ATCC CCL-1) were cultured in RPMI-1640 media (Gibco) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/ml) and streptomycin (100μg/ml). The cells were

maintained at 37 °C in a humidified atmosphere containing 5% CO2. 2.8. Polyplex preparation

PAMAM derivative in serum-free medium with equivalent volume of the plasmids (pRL-CMV or shRNA) in serum-free medium were mixed to prepare desired carrier/plasmid (C/P) ratio (wt/wt), followed by in- cubating for 20–30 min at room temperature to form the polyplexes.

2.9. Transfection assay with luciferase reporter gene

Cells were cultured in 96-well plates at a density of 1 × 10⁴cells per well and incubated at 37^∘C. After 24 h, polyplexes with or without ap- tamer were prepared at optimal C/P ratio. Branched PEI 25 kDa (C/P 0.8) was considered as positive control. 20μl of complexes (containing 200 ng pRL-CMV) were added into each well containing complete medium and incubated at 37 °C for 4 h. Then, the medium was replaced with 100μl of fresh FBS supplemented RPMI and incubated at 37 °C for 24 h. The luci- ferase activity in cell lysate was measured after 24 h by using promega luciferase assay kit on Luminometer (Brthlod Detection System, Pforzheim, Germany). The results are reported as mean ± SD, n = 3.

2.10. Cytotoxicity assay

The cytotoxicity of either polyplexes or polyplex–aptamer conjugates was evaluated using the MTT colorimetric assay. Briefly, the cells were cultured in 96-well plates at a density of 1 × 10⁴cells per well. After 24 h, the cells were treated with the same amounts of polyplexes used for transfection experiment and maintained at 37 °C for 4 h. After replacing medium with fresh complete RPMI, cells were cultured for an additional 24 h 20μl MTT reagent in PBS (5 mg/ml) was added to each well and incubated for 2 h. After removing the medium, 100μl dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals in each well.

Absorbance was read with an ELISA microplate reader (TECAN infinite M200, Switzerland) at 570/630 nm. The cell viability (%) relative to control wells containing cells not treated with polyplexes was calculated as [A] test/

[A] control × 100. The results reported as the mean ± SD of triplicate.

2.11. Western blot analysis for shRNA delivery

Cells were seeded at a density of 1.2 × 10⁵ cells/well in 6-well plates and incubated for 24 h shRNA transfection was carried out with polyplexes with or without aptamer (1.6μg shRNA per well). The re- maining of the transfection procedure was as described above.

Expression levels of Bcl-xL protein in the cell lysates was determined by Western blot analysis as follows:

72 h post transfection, cells were harvested and placed in homo- genization buffer (pH: 7.4) containing 50 mM Tris–HCl, 2 mM EGTA,

2 mM EDTA, 10 mM NaF, 0.2% sodium deoxycholate, 1 mM Na3VO4, 10 mMβ-glycerophosphate, 1 mM PMSF and 2μl complete protease in- hibitor cocktail. The cell lysate was sonicated on ice for 40 s using probe sonicator (Bandelin Sonoplus mini 20, Bandelin Electronics, Berlin, Germany) followed by measuring the protein content with BioRad protein assay kit. The equal samples of total proteins (10μg) were subjected to 10% SDS–PAGE and then blotted onto PVDF membranes (Millipore, Billerica, USA). The membranes were blocked in TBST (Tris-buffered saline with Tween 20) containing 5% skimmed milk at 4 °C for overnight.

Then, the blocked membranes were incubated with mouse anti-human Bcl-xL or beta actin antibody (cell signaling, 1:1000 dilution) for 2 h fol- lowed by incubation with HRP-labeled Rabbit anti-mouse secondary an- tibody (cell signaling, 1:3000 dilution) for 1.5 h at room temperature. The Bcl-xL bands were identified with enhanced chemiluminescence reagent (Pierce ECL western blotting substrate, USA) and Alliance Gel-doc (Alliance 4.7 Gel doc, UVtec UK). The intensity of protein bands was de- termined with UV Tec software (UK).

2.12. Apoptosis evaluation with annexin v/PI

After shRNA delivery to the cells with vector-aptamer-shRNA, cell apoptosis was investigated with annexin v/PI assay. Briefly, treated cells were trypsinized 48 h post transfection and centrifuged at 2500 rpm 4 °C.

Cells were resuspended in binding buffer 1X containing annexin V-FITC.

Following 15 min incubation in the dark at room temperature, cells were washed twice with binding buffer. Finally, cells resuspended in binding buffer containing PI and apoptosis was evaluated byflow cytometry in- strument (Becton Dickinson, California, USA) using the 488 nm laser for excitation. FITC was detected in FL-1 by a 525/30 BPfilter whereas PI was detected in FL-2 by a 575/30 BPfilter (BD Bio-sciences).

2.13. Statistical analysis

Results were analyzed using Student’st-test with GraphPad Prism version 6 software. P values ≤0.05 were considered as statistically significant. Data were reported as a mean ± SD of triplicates.

3. Results

3.1. Synthesized of modified PAMAMs vector

In our pervious study, synthesis and physicochemical characteriza- tions of different modified PAMAM vectors with different carboxyalkyl or carboxyalkyl-PEG chains were performed (Ayatollahi et al., 2015).

The vector including G4 [(10C-30%)(10C-PEG)] with high transfection efficiency and minimal cytotoxicity was selected for targeted delivery in this study.Scheme 1represents the brief overview of the synthesis of modified PAMAM vector.

Scheme 1.Modification of PAMAM by bromocarboxylic acids where n = 1, 5, 9, and 15 (Ayatollahi et al., 2015).

3.2. Aptamer binding to modified PAMAM

AS1411 aptamer was coupled to the selected vector either cova- lently or non-covalently. Covalent conjugation of aptamer to modified PAMAM was performed based on the formation of an amide bond. Non- covalent conjugation of negatively charged aptamer to modified PAMAM was based on electrostatic interactions.Scheme 2 illustrates the approach for covalent conjugation of modified PAMAM to aptamer.

3.3. Confirmation of aptamer binding to modified PAMAM

To verify the attachment of aptamer to modified-PAMAM vector, agarose gel electrophoresis with/without heparin was performed. Results indicated that heparin could release aptamer from the vector-Apt complexes; however the release of non-covalent aptamer from the vector was considerably more than covalent one. Image analysis of band (L3) by UV Band software re- vealed that about 66% of conjugated aptamers were covalently coupled to the vector through amid bond whereas the remaining aptamers (34%) were attached to the vector by electrostatic interactions (Fig. 1).

3.4. Particle size and surface charge of aptamer-polyplex conjugates

The particle size and zeta potential of polyplexes before/after binding to aptamer at optimal C/P ratio were determined by the Malvern Nano ZS instrument (Malvern Instruments, UK). These poly- plexes exhibited typical particle size between 128 and 230 nm. The size of polyplexes increased after binding to aptamers. Surface charge of all polyplexes even after aptamer conjugations were positive and between 12.76-19.13 mv (Table 1).

3.5. Immunocytochemistry for evaluation of nucleolin receptors on surface cells

Expression of target receptor on the surface of the selected cells was investigated by immunocytochemistry analysis as described above. The results confirmed the presence of nucleolin proteins on A549 cells but not on L929 cells as the negative control (Fig. 2).

3.6. Transfection efficiency

The transfection efficiency of polyplexes prepared from modified PAMAM and its aptamer conjugates was determined in nucleolin-ex- pressing A549 cells and L929 cell as negative control using Renilla lu- ciferase reporter gene (Fig. 3). Branched PEI 25 kDa at C/P 0.8 was also considered as positive control. Modified vector showed considerable higher transfection efficacy compared to PEI 25 kDa. Furthermore, this PAMAM derivative significantly improved the transfection efficiency even after covalent or non-covalent aptamer binding compared to the non-targeted vector in A549 cells and non-covalent conjugation of ap- tamer showed more efficiency compared to covalent binding.

Scheme 2.Covalent binding of aptamer to PAMAM derivative.

Fig. 1.Agarose-gel electrophoresis for confirming of covalent or non-covalent binding of aptamer to the vector, L1: Vector-Apt (non-covalent), L2: Vector−Apt (non-covalent) + heparin, L3: Vector −Apt (covalent) + heparin, L4: Vector−Apt (covalent), L5:

Aptamer.

Table 1

The particle size and zeta potential of polyplexes before/after aptamer conjugation with AS1411 aptamer at the optimum C/P ratio. Data are expressed as mean values ± SD.

Vector Size (nm) Zeta potential (mv)

G4 [(10C-30%)(10C-PEG)]-C/P 6 128 ± 1.9 13.67 ± 2.5 G4 [(10C−30%)(10C-PEG)]-C/P 6 Apt-phy^a 230 ± 2.4 19.3 ± 1.8 G4 [(10C−30%)(10C-PEG)]-C/P 6 Apt-cov^a 148 ± 3.01 12.76 ± 1.4

aApt-phy and Apt-cov were defined, respectively as physical (non-covalent) and covalent conjugations of aptamer to modified PAMAM.

Fig. 2.Immunocytochemistry analysis for the detection of target re- ceptors on A549 cells as positive cells. Surrounding colors of A549 cells indicated the presence of membrane receptors on the cell sur- faces.

3.7. Cytotoxicity assay

The cytotoxicity of polyplexes before/after conjugation to the ap- tamer was investigated in nucleolin positive and negative cells using MTT assay. PEI 25 kDa was used as control. As shown inFig. 4, all polyplexes exhibited more than 70% viability even after aptamer con- jugation.

3.8. In vitro gene silencing

The expression level of Bcl-xL protein was evaluated by Western blot analysis 72 h post-transfection with G4(10C-30%) (10C- PEG) conjugated aptamer (non-covalently) and shRNA plasmid.

Reduction in the Bcl-xL protein expression with vector-aptamer- shRNA was about 55% compared to vector-aptamer-scrambled shRNA (p < 0.001) in A549 cells. Treatment with vector and vector-aptamer

alone or scrambled shRNA did not significantly change the protein expression levels (Fig. 5). There is no reduction in Bcl-xL protein ex- pression when L929 cells were treated with vector-aptamer-shRNA (Fig. 6).These results confirmed the selective silencing of Bcl-xL ex- pression by shRNA and indicated that the conjugation of AS1411 ap- tamer to modified PAMAM could improve the Bcl-xL knockdown in comparison with modified PAMAM alone.

3.9. Annexin-V-FITC/PI staining for the detection of the apoptotic cells

Following Bcl-xL silencing with shRNA delivery, apoptosis in the target cells was investigated by the annexin V/PI assay. Consistent with our data from Western blot analysis which indicated the significant reduction in Bcl-xL levels in A549 cells, apoptosis and necrosis were evident in dot plots as shown inFig. 7. Flow cytometric analysis re- vealed that Bcl-xL silencing with vector-aptamer-shRNA after 48 h caused about 11% apoptosis more than vector-shRNA in A549 cells.

Fig. 3.Transfection activity of modified polymer with or without AS1411 aptamer and PEI 25 as gold standard in A549 as positive cells (A) and L929 as negative cells (B) using luciferase reporter gene. P values: P < 0.05 and P < 0.01 were considered as * and **, re- spectively (t-test). All assays were performed in triplicates and re- presented as mean ± SD.

Fig. 4.Cytotoxicity of polyplexes composed of modified polymer with or without AS1411 aptamer and luciferase reporter gene in A549 as positive cells (A) and L929 as negative cells (B) using MTT assay.

Fig. 5.Western blot analysis of Bcl-xL down-regulation after transfection with Bcl-xL shRNA and scrambled shRNA in A549 cells. V and apt were defined as G4(10C-30%) (10C-PEG) vector and aptamer, respectively.

Fig. 6.Western blot analysis of Bcl-xL silencing after transfection with Bcl-xL shRNA and scrambled shRNA in L929 cells as negative control. V and apt were defined as G4(10C- 30%) (10C-PEG) and aptamer respectively.

4. Discussion

RNAi-based gene therapy is emerging as an effective system for si- lencing of oncogenic or anti-apoptotic genes such as Bcl-xL. Bcl-xL, related to the Bcl-2 family, is a mitochondrial transmembrane protein and involved in tumorigenesis with inhibition of some tumor sup- pressors (Cory et al., 2003; Kirkin et al., 2004; Yip and Reed, 2008).

Two main systems for RNAi are the synthetic double-stranded small interfering RNAs (siRNAs) and the plasmid-based short hairpin RNAs (shRNAs). shRNA can be synthesized in the nucleus so low copy num- bers of shRNA provide more efficient, persistent and also less off-target gene knockdown compared to siRNA (Rao et al., 2009). Recently, sys- tems based on siRNA/shRNA have been developed for Bcl-2–selective inhibition (de Mello et al., 2017; Ebrahimian et al., 2017). Other in- hibitors including small molecules such as ABT-263 (Navitoclax) and

ABT-199 (Venetoclax) are in clinical trials for the treatment of solid tumors and hematological malignancies either with or without combi- nation with chemotherapeutics (Leverson et al., 2015).

For efficient delivery of shRNA to cancer cells, like other plasmids, various nano-carriers have been designed and used. Among different types of carriers, dendrimers with notable features are very attractive for biomedical application included gene/drug delivery. Dendrimers are monodispersed macromolecules with well-defined structure, size, shape and molecular weight and have symmetrical structures. Their high solubility in polar or non-polar solvent is related to hydrophilic or hydrophobic end groups (Abbasi et al., 2014; Agrawal and Kulkarni, 2015; Klajnert and Bryszewska, 2000). Dendrimers, especially PAMAM, have been extensively used for the delivery of genetic materials due to their unique characteristics. PAMAM possess high density of primary amino groups on the surface so can effectively bind, condense and Fig. 7.The study of apoptosis induced by A: vector [G4(10C-30%)(10C-PEG)], B: vector/scramble shRNA, C: vector/Bcl-xL shRNA, D: vector/aptamer, E: vector/aptamer/scramble shRNA, F: vector/aptamer/Bcl-xL shRNA, G: untreated cells (A549).

protect genetic materials. These complexes are called ‘dendriplexes’.

High density of amine groups in PAMAM structure which are proton- able under endosomal pH confer it the buffering effect and conse- quently facilitated endosomal escape (Yang et al., 2015). Some factors could influence on transfection efficiency of dendrimers such as mole- cular weight (dendrimer generation), structuralflexibility and surface charge of the complexes. With higher generation, transfection effi- ciency is improved but cytotoxicity is also increased [40]. Dendrimers with more structuralflexibility including triazine dendrimers exhibited enhanced DNA interactions and increased transfection efficiencies (Merkel et al., 2009; Merkel et al., 2011). In some studies, it was shown that PEG with hydrophilic and flexible nature could improve the properties of PAMAM/plasmid complex such as steric stability, prevent unwanted interactions with nearby molecules and reduce cytotoxicity (Luo et al., 2002; Qi et al., 2009). PAMAM modified with anionic group (carboxylate) displayed lower cytotoxicity than the cationic (amine) compounds [43]. In our pervious study, different carboxyalkylate- grafted PAMAM dendrimers (G4 and G5) with or without PEG mod- ification were synthesized and their cytotoxicity and transfection effi- ciency using two reporter genes (luciferase and EGFP) into cancerous cells were evaluated (Ayatollahi et al., 2015). In this study, in order to improve the transfection efficacy of PAMAM for targeted delivery of Bcl-xL shRNA into cancer cells, 10-bromodecanoic acid (10C) and 10C- PEG were conjugated to PAMAM (based on the best results obtained in the transfection and cytotoxicity assays in our previous study). The PEGylated formulation was selected in order to improve the delivery system properties for the future in vivo studies. Then anti-nucleolin aptamer (AS1411) was grafted to the PAMAM derivative covalently or non-covalently. In different studies, PAMAM functionalized with var- ious targeting ligands such as small molecules (Ke et al., 2009; Kumar et al., 2010), peptides (Huang et al., 2007), antibodies (Theoharis et al., 2009) and aptamers (Wang et al., 2015; Wu et al., 2014) have exhibited improvement in site specific gene/drug delivery and reduction in cy- totoxicity on normal cells. Li et al. used AS1411 aptamer conjugated to PEGylated cationic liposome for targeted delivery of anti-BRAF siRNA to melanoma cells. The results displayed specific knock down of proto- oncogene B-RAF (BRAF gene) in A375 cells and tumor xenograft mice (Li et al., 2014). In another study, Aravind et al. functionalized the PLGA-lecithin-PEG nanoparticles with AS1411 aptamer to deliver pa- clitaxel to cancer cells. The results indicated selective cytotoxicity in MCF-7 tumor cells (Aravind et al., 2012).

AS1411 is a DNA aptamer in Phase II of clinical trial (Pahle and Walther, 2016) which binds specifically to nucleolin that is highly ex- pressed on many cancer cells membrane including acute myelogenous leukemia (AML) (Mongelard and Bouvet, 2010), MDA-MB-231, A549, C6, HeLa (Dapic et al., 2002), AGS (Watanabe et al., 2010) and A549 (Ireson and Kelland, 2006). We confirmed nucleolin expression on the surface of A549 cells with immunocytochemistry analysis. The results revealed the presence of nucleolin on the A549 plasma membrane but not on normal cells.

Aptamer binding on the cell surfaces were evaluated withflow cy- tometry assay as well as the expression of nucleolin receptor on the surface of cancer cell was explored with immunocytochemistry ex- periment. The results confirmed the presence of nucleolin on A549 cells but not on L929 as control cells. Previous studies reported the over- expression of nucleolin on the surface of cancer cells (Kufe, 2009;

Watanabe et al., 2010).

Size and surface charge of nanoparticles play an important role in stability, cell interaction and uptake, cytotoxicity and transfection ef- ficacy (He et al., 2010; Rejman et al., 2004; Shahidi-Hamedani et al., 2013). When aptamer was conjugated to the vector, size and surface charge of particles increased. Increase in surface charge may be due to some changes in the physical structure after aptamer conjugation that lead to appearance of some positive charges from deep polymer layers.

However, both aptamer conjugations (covalent and non-covalent con- jugation) led to nanoparticles with appropriate size and suitable surface

charge which could interact with cell membranes.

Luciferase expression assay of aptamer-conjugated vectors revealed that G4 [(10C-30%)(10CPEG)] grafted non-covalently to AS1411 ap- tamer could efficiently deliver luciferase plasmid about 8 folds to A549 cancer cells compared to control cells (L929 cells). Cytotoxicity results exhibited that vector conjugated to aptamer (covalently or non-cova- lently) did not display any considerable toxicity on cells. Consequently, G4 [(10C-30%)(10CPEG)] vector conjugated non-covalently with AS1411aptamer was selected for targeted delivery of shRNA suppres- sing Bcl-xL mRNA. Overexpression of Bcl-xL, an anti-apoptotic protein of Bcl-2 family, has been reported in various cancers including neuronal tumors (Krajewski et al., 1997), breast cancer (Fiebig et al., 2006), adenocarcinoma (Krajewska et al., 1996), bladder cancer (Kirsh et al., 1998) and gastric cancer (Kondo et al., 1996). Bcl-xL contributes to tumorigenesis, tumor expansion and resistance to anticancer treatments (Fiebig et al., 2006; Kirkin et al., 2004). Hence, suppression of Bcl-xL like other BCL-2 proteins could be considered as a promising class of anticancer therapy. Different carriers and suppressing agents such as siRNA or shRNA were used for this purpose (Conlon et al., 2011; Kim et al., 2010).

To investigate the Bcl-xL silencing activity with shRNA using syn- thetized vectors, we used Western blot analysis. Our results indicated that G4[(10C-30%)(10CPEG)]-Apt could significantly down regulate the Bcl-xL expression in A549 cells (about 55% compared to scrambled shRNA) while there was no decrease in Bcl-xL expression in normal L929 cells. Furthermore, apoptosis induced in cancer cells with targeted delivery of shRNA was identified using Annexin V-FITC/PI assay.

Results after 48 h of transfection indicated that shRNA could induce apoptosis (late apoptosis) about 14% more thanscramble plasmid. Our results supported the fact that efficient and targeted apoptosis in cancer cells could be induced by functionalized PAMAM-aptamer conjugate through knockdown of Bcl-xL expression using shRNA.

5. Conclusions

In this study, we designed and developed novel targeted delivery system based on modification of PAMAM with10-bromodecanoic acid (10C) and 10C-PEG. PEGylation of the vector improved its properties for in vivo studies. AS1411 aptamer conjugated vector-shRNA com- plexes could efficiently target nucleolin positive cells (A549 cells), knockdown the expression of Bcl-xL protein and induce apoptosis in cancer cells. Our results indicated that the developed targeted nano- complex in this study could be used in further studies for gene therapy specially for pulmonary systems with great features including high transfection efficiency, low cytotoxicity and selective inhibition of cancer cells.

Acknowledgments

This work was funded by the Mashhad University of Medical Sciences, Mashhad, Iran (grant number: 89189). We acknowledge the financial support from Iran Nanotechnology Initiative. We would like to gratefully acknowledge Dr. Sara Amel Farzad at Avicenna Research Center for her assistance with laboratory experiments.

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