Single-walled carbon nanotubes functionalized with aptamer and piperazine-polyethylenimine derivative for targeted siRNA delivery into breast cancer cells.

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Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Pharmaceutical nanotechnology

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Single-walled carbon nanotubes functionalized with aptamer and piperazine–polyethylenimine derivative for targeted siRNA delivery into breast cancer cells

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Marzieh Mohammadi a,1, Zahra Salmasi a,1, Maryam Hashemi b , Fatemeh Mosaffa c , Khalil Abnous a, **, Mohammad Ramezani a, * a

Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, P.O. Box 91775-1365, Iran Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, P.O. Box 91775-1365, Iran c Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, P.O. Box 91775-1365, Iran b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 December 2014 Received in revised form 7 February 2015 Accepted 10 February 2015 Available online xxx

Epithelial cell adhesion molecule (EpCAM) is a glycosylated type 1 membrane protein which is frequently over expressed in most solid tumors and it has recently been identified as a cancer stem cell (CSC) marker. Specific targeting of CSCs using nano-carriers would enhance treatment efficacy of cancer. In this study, we used a RNA aptamer against EpCAM (EpDT3) attached physically to our newly synthesized non-viral vector, based on single-walled carbon nanotube (SWNT) conjugated to piperazine–polyethylenimine derivative. The DNA transfection efficiency and siRNA delivery activity of the synthesized vector was investigated against upregulated BCL9l, which has been associated with breast and colorectal cancers. The complexes of the vector–aptamer/siRNA could specifically induce apoptosis by more than 20% in MCF-7 cell line as a positive EpCAM than MDA-MB-231 cells which are EpCAM negative. The decrease of BCL9l protein level was observed with western blot analysis in MCF-7 cells indicating the targeted silencing activity of the complex. ã 2015 Published by Elsevier B.V.

Keywords: Gene delivery Aptamer Epithelial cell adhesion molecule Single-walled carbon nanotube siRNA

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1. Introduction After heart disease, cancer is the second leading cause of mortality. Breast cancer is the second most common cause of death in women after lung cancer (Siegel et al., 2013). Many investigations have been performed to overcome cancer cells resistance against chemotherapeutic agents. Amongst the promising approaches which have been emerged to fight against cancer, gene therapy has gained dramatic interest (Merdan et al., 2002; El-Aneed, 2004). It has been demonstrated that small interfering RNAs (siRNA) have a great potential as nucleic acid therapeutics because of their potent and specific RNAi triggering activity (Li et al., 2013a). However, due to the immunogenicity and

* Corresponding author. Tel.: +98 51 37112471; fax: +98 51 37112470. ** Corresponding author. Tel.: +98 51 38823251; fax: +98 51 38823255. E-mail addresses: [email protected] (K. Abnous), [email protected] (M. Ramezani). 1 These authors contributed equally to this work.

safety problems of viral vectors, the most serious challenge in gene therapy is developing a safe and efficient non-viral carrier system Q2 for transferring the genetic materials into the target cells. Polyethylenimine (PEI) is one of the most commonly used polycations with good DNA condensing properties. PEI with branched structure contains primary, secondary and tertiary amines which can be protonated and provide buffering capacity over a wide range of pH. This effect facilitates endosomal release of the PEI–nucleic acid complex into the cytosol before degradation by lysosomal nuclease. The increase in transfection efficiency of PEI is directly related to its molecular weight. However, high molecular weight PEIs lead to dramatic toxicities (Boussif et al., 1995; Dehshahri et al., 2009; Parhiz et al., 2013) which might be as a result of their strong positive surface charge (Moghimi et al., 2005; Song et al., 2010). Previous studies proved that applying hydrophobic modifications to PEI by alkylcarboxylation of PEI would result in improved transfection efficiency and lower cytotoxicity (Dehshahri et al., 2009; Oskuee et al., 2010; Parhiz et al., 2013). This lower cell toxicity may be due to the reduction of the overall positive surface charge of the particle and consequent

http://dx.doi.org/10.1016/j.ijpharm.2015.02.031 0378-5173/ ã 2015 Published by Elsevier B.V.

Please cite this article in press as: Mohammadi, M., et al., Single-walled carbon nanotubes functionalized with aptamer and piperazine– polyethylenimine derivative for targeted siRNA delivery into breast cancer cells. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j. ijpharm.2015.02.031

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lower interaction between the polyplex and cell membrane which is the main cause of membrane damage (Moghimi et al., 2005; Song et al., 2010). Single-walled carbon nanotubes (SWNTs) are other valuable candidates which have been used for siRNA delivery. Their unique properties including nano-needle structure enable them to easily cross the plasma membrane via endocytosis-independent pathway (Lacerda et al., 2007; Kesharwani et al., 2012). However, their poor solubility and little biocompatibility have limited their use as an efficient carrier. One approach to facilitate their solubilisation is to introduce positively charged groups onto SWNTs surface. Such functionalized CNTs are not only soluble in water but also they can bind electrostatically to negatively charged molecules such as siRNA or DNA (Wu et al., 2008; Varkouhi et al., 2011). Targeted delivery of therapeutics could be a promising approach for the delivery of the gene to specific cell populations while decreasing cytotoxicity and improving the efficiency of gene therapy. Aptamers are single-stranded, synthetic DNA or RNA molecules which specifically recognize and bind tightly to their targets due to their secondary or tertiary structure. This high affinity enables aptamers to directly bind to specific surface receptors. Thus, it is believed that nanoparticle–aptamer conjugates may enhance the efficiency of the delivery and decrease the systematic toxicity and immunogenicity (Zhou et al., 2012; Li et al., 2013b). Epithelial cell adhesion molecule (EpCAM) is a glycosylated type 1 membrane protein which is frequently overexpressed in most solid tumors and it has recently been identified as a cancer stem cell marker (CSCs) (Shigdar et al., 2011; Subramanian et al., 2012). CSCs are a rare number of cells within the tumor responsible for tumor initiation, self-renewal, relapse and metastasis. In fact, EpCAM overexpression in breast cancers is associated with drugresistance and poor overall survival (Osta et al., 2004). Recently, a 19-nt RNA aptamer against EpCAM was isolated using cell surface SELEX (systematic evolution of ligands by exponential enrichment) and it was used as a target-specific delivery of doxorubicin to EpCAM-positive cells (Shigdar et al., 2011; Subramanian et al., 2012). It was assumed that specific targeting of CSCs using nano-carriers will enhance treatment efficacy of cancer. Canonical Wnt signaling pathway plays a central role in development and tissue renewal where mutations in this pathway is associated with colorectal and other cancers (Fodde and Brabletz, 2007). BCL9l up-regulation in breast and colorectal cancer cells is required for enhanced Wnt signaling and increased proliferation and metastatic behavior of tumor cells (Fodde and Brabletz, 2007; Deka et al., 2010). In the current study, EpDT3 aptamer was physically attached to a non-viral vector based on single-walled carbon nanotubes conjugated to a derivative of polyethylenimine and used for targeted siRNA delivery against BCL9l.

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2. Material and methods

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SWNTs and propidium iodide were purchased from Sigma (Germany). Branched polyethylenimine (PEI; average MW 25 kDa) was purchased from Sigma–Aldrich (US). Ethidium bromide was obtained from Cinnagen (Tehran, Iran). 7-Hydroxy benztriazole hydrate (HOBt) and 1-ethyl-3-[3-dimethylaminopropy]carbodiimide hydrochloride (EDC) were purchased from Fulka (Germany). The plasmid pRL-CMV-luc, Renilla luciferase assay kit and Cell Titer 961 Aqueous One Solution Cell Proliferation assay (MTT) were obtained from Promega (Madison, USA). EpCAM aptamer (50 -G5GA56GG66A555GG65G-30 ) was purchased from Microsynth (Switzerland). BCL9l siRNA (antisense seq.: GGAUGUUCAAGAUCCCAUGCAGCTC) was purchased from Origene, USA.

2.1. Cell lines and cell culture

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MCF-7 and MDA-MB-231 (human breast adenocarcinoma) cell lines were purchased from the Pasteur Institute of Iran and cultured at 37 C, 5% CO2 atmosphere in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco).

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2.2. Preparation of carboxylated SWNTs

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SWNTs (2 mg) was dispersed in HNO3 solution (30 ml, 2.5 M) and sonicated for 1 min; then, refluxed for 24 h at 95 C while stirring. After sonication for 30 min, reaction was refluxed for additional 48 h. The acid-treated SWNTs were filtrated and washed extensively in deionized water until a constant pH value in the range of 6–7 was obtained. Then filtrated SWNTs were collected and desiccated for 24 h at 50 C.

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2.3. Preparation of SWNT–PEI–piperazine conjugate

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Polyethylenimine (PEI)–piperazine conjugate was prepared using 7-hydroxy benzotriazole hydrate (HOBt) and 1-ethyl-3-[3dimethylaminopropy]carbodimide hydrochloride (EDC) as coupling agents. Briefly, PEI 25 kDa (50 mg) was dissolved in distilled aqueous HOBt solution (14.1 mg) and was added drop-wise to the gently stirred solution of piperazine-2-carboxylic acid dihydrochloride (14.5 mg) and EDC (20 mg) in distilled water. The reaction was allowed to proceed for 24 h. The final product was dialyzed against distilled water using Spectra/Por dialysis membrane (6000–10,000 Da cut-off, Spectrum Laboratories, Houston, USA) for 3 days to remove the unreacted materials and then lyophilized. Amide bond formation was confirmed by Fourier transform infrared spectroscopy (FTIR, PerkinElmer Spectrum Two, US). The degree of substitution of primary amines on PEI was determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay as described previously (Snyder and Sobocinski, 1975). In the next step, carboxylated nanotubes were dispersed in 3 ml of water and sonicated for 2 min. To activate the carboxylate groups for amide bond formation, EDC (1.5 mg) was added to the dispersed CNT solution followed by sonication for 2 min, and then stirred for 1 h. HOBt (1 mg) was added to PEI–piperazine solution and sonicated for 3 min, then was added drop-wise to carboxylated SWNT mixture. The reaction was allowed to proceed for 4 days at room temperature followed by dialysis against water to remove the unreacted materials. Final product was lyophilized and FTIR spectroscopy was used to cofirm amide bond formation between carboxylated CNT and PEI–piperazine.

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2.4. Particle size and zeta measurements

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The hydrodynamic diameter and zeta potential of the polyplexes (vector/pDNA or vector/pDNA/aptamer) were determined using Malvern ZS Nano Instrument (Malvern Instruments, UK) in a salt-free buffer (20 mM HEPES, 5% glucose, pH 7.4).

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2.5. DNA binding assay

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The ability of the vector to condense DNA was evaluated by ethidium bromide (EtBr) exclusion assay and determination of fluorescence intensity (lexcitation: 510 nm and lemission: 590 nm) using the Jasco FP-6200 spectrofluorimeter (Jasco Global, Hachioji, Tokyo, Japan). Briefly, PEI, synthetized vector or vector–aptamer in HBG buffer (20 mM HEPES in 5% aqueous glucose solution, pH 7.4) was added step wise to solution of the EtBr (400 ng/ml) and pDNA (20 mg/ml) in HBG buffer. Then fluorescence intensities were recorded. The fluorescence intensity of the EtBr plus pDNA was considered as 0% condensation and the fluorescence intensity of

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EtBr without pDNA was taken as 100% condensation. Results are reported as mean S.D. (n = 3).

Germany). Results were reported as relative light units (RLU) per number of seeded cells.

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2.6. Physical conjugation of aptamer to the vector

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The SWNT–PEI–piperazine vector was added to the aptamer at 5% w/w (aptamer/vector) in nuclease-free water and incubated for 2 h at room temperature while stirring.

MCF-7 and MDA-MB-231 cells were seeded at 104 cells/well in 96-well plates and cultured overnight. Then, cells were treated with the same amounts of polyplexes used for transfection experiments and maintained at 37 C for 4 h. After replacing medium with fresh FBS-supplemented RPMI (100 ml), cells were incubated for an additional 24 h. MTT reagent (20 ml, 5 mg/ml in PBS) was added to each well and incubated at 37 C for 1.5 h. After removing the medium, DMSO (100 ml) was added to each well to solubilize the formazon crystals and absorbance was measured by ELISA plate reader (TECAN infinite M200, Switzerland) at 570/630 nm. The cell viability (%) relative to control wells containing cell culture medium without polyplexes was calculated by [A]test/[A]control 100.

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2.12. siRNA transfection

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Cells were transfected using vector/siRNA complexes at the ratios of 2:1 and 4:1 w:w. 104 MCF-7 cells per well were seeded in 48-well plates one day prior to transfection assay. Cells were incubated for 24 h at 37 C under CO2 (5%) atmosphere. Then, the medium was removed and replaced with serum free Optimem medium (200 ml). 40 ml of complexes (equivalent to 10 nM BCL9l siRNA) were added into each well. After 6 h, the media were replaced with fresh serum supplemented RPMI and incubated at 37 C for 48 h. Then, the apoptotic cells were analyzed with propidium iodide (PI) staining.

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2.13. Analysis of apoptosis with propidium iodide staining

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Cells were trypsinized and centrifuged at 5000 g at 4 C for 15 min. Then, cells were re-suspended in hypotonic solution (300 ml) containing PI (50 mg/ml) in sodium citrate (0.1%) and Triton X-100 (0.1%). RNase A (100 mg/ml) also was added to degrade RNA and to prevent PI staining of RNA. Cells were then incubated for 2 h at room temperature followed by centrifugation at 5000 g for 15 min. The pellets were re-suspended in DPBS solution. The cells were analyzed by FACS calibur flowcytometer (Becton Dickinson, California, USA) using FL-2 channel (orange-red fluorescence: 585/42 nm).

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2.14. Western blot analysis

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MCF-7 cells were seeded at a density of 3 105 cells/well in 6 well-plates and incubated for 24 h at 37 C and 5% CO2. Prior to transfection, the medium was replaced with serum and antibiotic free medium (Optimem). After 20 min, polyplexes consisted of BCL9l siRNA (10 nM) and scrambled siRNA were added to each well. After 6 h, the medium was replaced with fresh serum-supplemented RPMI and incubated at 37 C for 48 h. Then, the medium was removed and the MCF-7 cells were trypsinized and washed with cold DPBS. Pellets were suspended in lysis buffer (100 ml) containing Tris–HCl (50 mM, pH 7.4), NaCl (150 mM), Triton-X 100 (1%, w/v), EDTA (1 mM), SDS (0.2% w/v), protease inhibitor cocktail (1%, w/v), phosphatase inhibitor cocktail (1%, w/v), and phenylmethyl sulphonyl fluoride (1 mM) and freezed at 80 C for 30 min. Pellets were centrifuged at 14,000 g at 4 C for 20 min. Then, the protein concentration was determined using Bradford protein assay kit (Bio-Rad, USA). 20 mg of total protein were loaded and separated on 8% acrylamide gel (Bio-Rad, USA). Then, the proteins were transferred to a PVDF membrane (Bio-Rad, USA). Membrane was blocked with 5% (v/v) skim milk in TBST (10% Tris-buffered

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2.7. Preparation of polyplexes

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The SWNT–PEI–piperazine vector and vector–aptamer conjugate were diluted in HBG buffer and incubated with plasmid DNA (equivalent to 200 ng pRL-CMV) for 20 min at room temperature.

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2.8. Evaluation of vector–aptamer conjugation

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To confirm stable physical conjugation of the aptamer to the vector, the vector–aptamer, vector–aptamer–pDNA at C/P ratio 6 incubated with heparin (2 units) for 5 min. After addition of loading dye, the samples were run on 1% agarose gel in TAE (40 mM Tris/HCl, 1% acetic acid, 1 mM EDTA, pH 7.4) at 80 V for 90 min, followed by imaging using Alliane 4.7 gel doc (UK). To evaluate the stability of the vector–aptamer and vector–aptamer–pDNA complex, we studied the release of the aptamer from the complex at different times. The released aptamer was evaluated by gel electrophoresis as described in previous step.

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2.9. Binding assay of EpCAM aptamer

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To confirm binding of 30 FAM-labeled EpCAM aptamer, MCF-7 and MDA-MB-231 cells were detached by trypsin and aptamer binding affinity toward cells were analyzed as previously described (Shigdar et al., 2011). Briefly, cells were centrifuged at 1000 g for 5 min and re-suspended in MgCl2 (5 mM) solution. Then, 5 105 cells were enumerated and again centrifuged at 1000 g for 5 min. The pellet was re-suspended in assay buffer containing DPBS supplemented with MgCl2 (0.1 mg/ml), sodium azide (0.2% w/v) and FBS (5%). The blocking step was carried out at 4 C for 1 h in the assay buffer followed by the re-suspension of the cells in the binding buffer (DPBS supplemented with MgCl2 (2.5 mM), sodium azide (0.2% w/v)). Then, FAM-labeled aptamer (1 ml, 1 mM) was added and the mixture was incubated on ice while shaking for 1 h. Thereafter, cells were centrifuged and re-suspended in MgCl2 (2.5 mM) solution 3 times and finally suspended in binding buffer and analyzed by FACScalibur flow cytometer (Becton Dickinson, California, USA) using FL-1 channel. Data were analyzed using WinMDI 2.9 software.

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2.10. Cell transfection with luciferase reporter gene

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Transfection studies were performed to determine efficiency of the synthesized vector in both MCF-7 and MDA-MB-231. Cell lines were seeded at a density of 104 cells/well and incubated in RPMI supplemented with 10% fetal bovine serum for 24 h. PEI 25 kDa at C/P 0.8 was used as positive control. Polyplexes were prepared at different C/P ratios (weight:weight ratio of vector/plasmid) ranging from 2:1 to 6:1 in HBG buffer. The polyplex mixtures containing 200 ng pDNA with or without aptamer were added to each well and incubated at 37 C for 4 h. Then, the medium was removed and replaced with fresh FBS supplemented RPMI (100 ml) and incubated at 37 C for 24 h. Lysis buffer (Promega, Madison, WI) was added to each well and the luciferase activity was measured using Promega Renilla assay kit (Promega, Madison, WI, USA) and a luminometer (Berthold Detection Systems, Pforzheim,

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saline (TBS) 10X, 0.1% Tween 20), for 3 h at room temperature and washed 3 times in TBST each time 5 min. Membranes were probed with BCL9l rabbit polyclonal antibody (Abcam, UK) diluted in TBST (1:1000) for 2 h. After 3 washes with TBST, membranes were probed with rabbit IgG secondary antibody (1:2000) (Cell Signaling, USA). Bands were visualized by using Enhanced Chemiluminescence (ECL) kit (Bio-Rad, USA) and Alliane 4.7 gel doc (UK). Protein bands were analyzed using UVIband software (UVtec, UK).

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2.15. Statistics

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Student’s t-test was performed for statistical comparisons. Data are presented as mean S.D. P values 0.05 were considered as statistically significant.

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3. Results

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3.1. Synthesis and characterization of the SWNT–PEI–piperazine conjugate

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To synthesize SWNT–PEI–piperazine (vector), 30% of the primary amines of the PEI 25 kDa were modified by attaching to piperazine-2-carboxylic acid. The degree of amine grafting, as measured by TNBS assay, was 26.7% mol. The conjugation of

piperazine moiety to PEI structure was evaluated by comparing FTIR spectra before and after conjugation. Formation of amide bond was confirmed by the appearance of band around 1630–1690 cm1 corresponding to carbonyl group. Carboxylated SWNTs were attached to PEI–piperazine derivative via amide bond formation. The formation of the final product was confirmed by FTIR spectrum in which signals corresponding to carbonyl groups of functionalized CNT (1719 cm1, 1627 cm1) and piperazine-2-carboxylic acid (1756 cm1) were disappeared while a new carbonyl signal was appeared at 1654 cm1 (Fig. 1). As it is shown in Fig. 2a, the aptamer formed stable conjugate with the vector as it remained condensed after seven days as evident by gel electrophoresis. However, addition of heparin to the vector–aptamer and vector–aptamer–pDNA complexes led to the release of both pDNA and the aptamer from the vector (Fig. 2b). Particle size and surface charge of the vector were obtained at C/P ratio of 6 (the C/P ratio with highest transfection efficiency in MCF-7 cells) and were compared to PEI 25 kDa as the control. Also we examined the particle size and zeta potential of vector conjugated to the aptamer. The surface charge of the polyplexes were positive and there was no significant difference between the size of the SWNT–PEI– piperazine vector (85.08 nm) and that of the PEI 25 kDa (78.17 nm), as a control (P > 0.05). However, the size of the vector conjugated to aptamer increased to 245.3 nm.

Fig. 1. Fourier transform infrared spectroscopy analysis of (A) f-SWNTs, (B) PEI 25 kDa, (C) piperazine-2-carboxylic acid dihydrochloride, (D) PEI–piperazine, and (E) final product. Peaks observed at 1750 cm1 and 1627 cm1 indicate that —COOH groups had been successfully grafted onto the surface of the SWNTs. Amide bonds between PEI and piperazineare shown in 1643.82 cm1 and in the final product new amid bonds were formed in 1654 cm1.


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Fig. 2. Stability of aptamer–vector conjugation by gel electrophoresis. (A) Evaluating stable aptamer conjugation to the vector at different times. (B) Agarose gel electrophoretic analysis of aptamer–vector conjugation after incubation with heparin.

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3.2. DNA binding assay

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Condensation of pDNA into nano-sized particles is an essential requirement for efficient delivery of DNA to the cells. Condensation of plasmid by the SWNT–PEI–piperazine vector was evaluated by the ethidium bromide (EtBr) exclusion assay. The fluorescence intensity of EtBr considerably increases upon intercalation with nucleic acid. By adding the aptamer conjugated vector, the DNA gradually binds to the complex so the intercalated EtBr is displaced, resulting in a significant reduction in the fluorescence intensity. Results of EtBr exclusion assay indicated that the complex was able to efficiently condense DNA and there was no significant difference between the condensation efficacies of the aptamer conjugated vector and PEI at C/P ratio of 0.5 and above (Fig. 3).

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Fig. 3. Plasmid DNA condensation of PEI 25 kDa and vector–EpDT3 aptamer measured by EtBr exclusion assay.

3.3. Determination of gene delivery efficiency of SWNT–PEI– piperazine vector

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Renilla luciferase assay kit was used to investigate the transfection activity of SWNT–PEI–piperazine vector in MCF-7 cell culture. Polyplexes were made in the C/P ratio of 2–6 and compared to the standard control, PEI 25 kDa, at C/P ratio of 0.8. The results revealed that the highest level of transfection efficiency was achieved at C/P ratio of 6 and the transfection efficiency was more than 10 folds higher than PEI 25 kDa (Fig. 4).

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Fig. 4. Transfection efficiency of unmodified PEI and SWNT–PEI–piperazine vector complexed with plasmid DNA at C/P ratios of 2, 4 and 6 in MCF-7 cell cultures (the results are reported as mean SD, n = 3).


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Fig. 5. Cellular viability of unmodified PEI 25 and SWNT–PEI–piperazine vector complexed with plasmid DNA at C/P ratios of 2, 4 and 6 in MCF-7 cell cultures (the results are reported as mean SD, n = 3).

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The cytotoxicity of the synthesized vector was evaluated by MTT assay. PEI 25 kDa was used as positive control. The results indicated that the vector was not significantly toxic to MCF-7 cells up to C/P ratio of 6 (Fig. 5).

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3.4. Aptamer binding to EPCAM positive cells

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To evaluate the expression of EpCAM on the selected cell lines, the FAM-labeled EpDT3 aptamer was incubated with EpCAM positive (MCF-7) and EpCAM negative (MDA-MB-231) cell lines and analyzed by flow cytometry. As shown in Fig. 6, the aptamer binds to both cell lines, however, MCF-7 (G mean: 13.18) has an obvious right shift in the geometric mean (G mean) fluorescence intensity compared to MDA-MB-231 (G mean: 5.31).

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3.5. Aptamer-conjugated vector luciferase transfection

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To evaluate the aptamer-conjugated vector transfection efficacy, MCF-7 and MDA-MB-231 cell lines were transfected at C/P ratio 6 (the ratio with the highest transfection efficiency of SWNT–PEI–piperazine vector). As shown in Fig. 7, transfection efficiency in the presence of EpCAM aptamer significantly increased in MCF-7 cell line compared to MDA-MB-231 (P < 0.001). Moreover, the transfection efficiency in aptamer conjugated vector/pDNA complex was 4 folds higher than vector/pDNA complex in MCF-7 cell line as EpCAM positive cells, while there was no significant change in the transfection efficiency in MDA-MB-231 as negative EpCAM cells (Fig. 7). MTT cytotoxicity assay showed that no significant cytotoxicity was induced by aptamer-conjugated vector in both EpCAM positive and EpCAM negative cell lines (Fig. 8).

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3.6. Analysis of apoptosis with propidium iodide staining

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The percentage of apoptotic cells following treatment with BCL9l siRNA was measured by PI staining and flow cytometry in order to detect fragmented DNA by analyzing the sub-G1 peak. As shown in Fig. 9, the SWNT–PEI–piperazine vector induced a concentration-dependent cytotoxic effect. The vector was cytotoxic at the C/P ratio 6 after 48 h; therefore, the optimum C/P ratios for siRNA delivery were determined at lower C/P ratios (2 and 4). Conjugation of the vector to EpCAM aptamer increased apoptosis in EpCAM positive cells (MCF-7) as compared to EpCAM negative cells (MDA-MB-231) (Figs. 9 and 10).

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3.7. Western blot analysis

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Western blot analysis of the MCF-7 cells transfected with siRNA against BCL9l was performed to determine BCL9l protein level. As indicated in Fig. 11, the protein level in the cells treated with vector–aptamer/siRNA polyplex decreased as compared to the vector–aptamer/scrambled siRNA polyplex.

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4. Discussion

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PEI is an effective non-viral vector with excellent properties such as efficient binding to cell surface and cellular uptake. However, it suffers from several disadvantages such as release of the polyplexes (PEI–DNA) from endocytic vesicles through osmotic burst mechanism which is the rate-limiting step in PEI transfection, cell toxicity with high molecular weight PEI and less effectiveness in siRNA applications (Boussif et al., 1995; Zintchenko et al., 2008; Parhiz et al., 2013). In this study, PEI–piperazine 30% was selected and coupled to the functionalized SWNTs. In the next step, the SWNT–PEI– piperazine nanoparticles were physically attached to EpCAM aptamer. These modifications were designed to investigate the changes in physiochemical and bioactivity of the formulation, aiming to improve DNA and siRNA transfection efficiency of SWNT-based gene carriers. Osmotic burst is result of buffering capacity in the endosomal pH range 5.0–7.2, which causes extra pumping of protons before fusion of the endocytic vesicle can take place with primary lysosomes. Extra pumping of protons results in extra counter ions and extra water causing an increase of osmotic pressure inside the endosome, which is responsible for the vesicle lysis. Therefore, it seems that increasing buffering capacity of PEI in the pH range 5.0–7.0 would increase transfection activity by facilitating the endosomal release. The effect of histidine on buffering capacity (Swami et al., 2007; Patnaik et al., 2012), and on fusogenic activity (Wang and Huang, 1984; Abe et al., 1985) on endosomal membranes in vectors have been evaluated. It was expected that piperazine with pKa value in the endosomal pH range, could improve the buffering capacity and transfection efficiency of the PEI derivative. SWNTs are a class of nanoparticles with unique properties such as the ability to easily cross the cell membrane, but their poor solubility and biocompatibility have limited their use as an efficient carrier (Madani et al., 2012). Toxicological studies have shown that introducing polar groups onto their surface facilitates SWNT solubility, improves their serum stability and makes them less toxic (Krajcik et al., 2008; Liu et al., 2009; Varkouhi et al., 2011). Thus in this work, we functionalized SWNTs by oxidation of some of the surface carbons to —COOH groups making them suitable for conjugation to the amine groups of PEI through amide bond formation. FTIR was used to evaluate successful functionalization of the SWNTs and to explore amide bond formation between SWNTs and PEI (Fig. 1). The transfection efficiency of SWNT–PEI–piperazine vector was significantly higher than the control (PEI 25 C/P 0.8) as shown in Fig. 4. There are other studies using cationic PEI grafted CNTs which reported successful gene delivery (Dong et al., 2011; Wang et al., 2013). In another study, PEI grafted multi walled carbon nanotubes (MWCNTs) immobilized pDNA on the surface of CNTs and enhanced the transfection efficiencies several times higher than PEI alone (Akinc et al., 2005). A probable explanation for the enhancement in the transfection level was the covalent conjugation of PEI to SWNTs as they have the ability to translocate directly into cytoplasm of target cells because of their nano-needle structure (Pantarotto et al., 2004; Cai et al., 2005). Moreover, the poor solubility and low biocompatibility of the SWNTs was overcome by introducing carboxyl groups onto their surface and

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Fig. 6. Flow cytometry histogram showing aptamer binding to EpCAM cell surface receptors in MCF-7 and MDA-MB-231 cell lines (EpCAM positive and EpCAM negative cell lines, respectively).

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providing a functional group for amide bond formation with primary amines of PEI 25 kDa. It has been shown that the primary amines of PEI responsible for more toxicity than secondary or tertiary amines (Abdallah et al., 1996). It is expected that substitution of some surface primary amines of PEI could result in reduced cytotoxicity. When piperazine and CNT were conjugated to PEI, primary amines reduced by 30% and the cell viability remained about 90% which is similar to PEI at C/P 0.8 (Fig. 5) but the cell viability improved significantly compared to PEI C/P 6 (data not shown). Particle size and surface charge of the synthesized nanoparticle was determined by adding pDNA to carrier at C/P ratio of 6 (the C/P ratio with high transfection efficiency in MCF-7 cells). At this C/P ratio, particles were in nanometer size range and were positively charged (Table 1). It has been proved that the uptake efficiency of

particles smaller than 100 nm was significantly higher than microparticles (Panyam and Labhasetwar, 2012). Electrostatic repulsion between positively charged polyplexes prevents them from aggregation. This positive charge improves the association between the polyplexes and the negatively charged cell membrane proteins (Gabrielson and Pack, 2006). Also it facilitates condensing DNA into nanosized particles (Parhiz et al., 2013). In order to develop an effective gene carrier with reduced side effects, it is essential to deliver the therapeutic agents selectively to their target sites. We used EpCAM RNA aptamer, as a targeting agent, to specifically deliver the genetic material to EpCAM positive cells. EpCAM overexpression is associated with metastasis and poor overall survival (Osta et al., 2004). Recent researches proved that EpCAM expression has a direct influence on cell proliferation, differentiation and metastasis(Osta et al., 2004; Munz et al., 2009).


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Fig. 7. Transfection efficiency of the vector and vector–aptamer conjugate complexed with plasmid DNA at C/P ratio of 6 compared to PEI 25 kDa at C/P ratio of 0.8 determined in triplicate in MCF-7 and MDA-MB-231 cell cultures in 96-well trays (p value < 0.001***, P > 0.05 ns).

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Thereafter, EpCAM antibodies have been extensively used to treat patients with EpCAM-positive tumors. Catumaxomab is a trifunctional EpCAM antibody which has been approved by European Commission for the treatment of EpCAM positive tumors (Gostner et al., 2011). However, clinical trials revealed that the use of EpCAM antibodies does not provide a remarkable clinical response. It is believed that the large size of the monoclonal antibody (mAb) restricts its distribution throughout the body (Shigdar et al., 2011).

Another important disadvantage is the immunogenicity triggered by the antibodies. EpCAM is a transmembrane glycoprotein and is universally expressed on the cells of epithelial origin and it has recently been identified as a cancer stem cell marker (CSCs) in breast, colorectal, pancreas, hepatic cancers and retinoblastoma (Yamashita et al., 2007; Munz et al., 2009; Shigdar et al., 2011; Subramanian et al., 2012). MDA-MB-231 is a low-expressing EpCAM cell line with mesenchyme-like phenotype. However, EpCAM is overexpressed in cell lines such as T47D, MCF-7, and SkBr3 that are cancer cells with epithelial morphology (Martowicz et al., 2012). We showed that EpDT3 aptamer could efficiently bind to EpCAM receptor which was overexpressed on MCF-7 cell line (Fig. 6). It is believed that aptamer–nanoparticle conjugates may enhance the efficiency of the delivery and decrease the systematic toxicity and immunogenicity of drugs (Zhou et al., 2012; Li et al., 2013b). Thus, SWNT–PEI–piperazine/DNA complex was physically bound to the EpDT3 aptamer and its transfection efficiency was evaluated. By conjugating aptamer to the vector, the size of the polyplex significantly increased but the overall surface charge of the particle remained positive (Table 1). As indicated in Fig. 6, the EpCAM aptamer demonstrated higher affinity and selectivity for MCF-7 cells (EpCAM positive) over MDA-MB-231 cells (Chen et al., 2012). Studies have demonstrated that EpCAM aptamer binds to the cell surface receptor and induce receptor-mediated endocytosis (RME). Internalization is crucial for a cancer-targeting ligand to ensure the complete delivery and release of the therapeutic agents inside the cell. Higher accumulation of the therapeutic agents inside the cells helps to overcome the multi-drug resistance (Shigdar et al., 2011). The data obtained indicated that physical conjugation of the EpDT3 aptamer to the pre-formed polyplex (SWNT–PEI–piperazine/pDNA complex) could enhance the transfection efficiency up to 7 folds in MCF-7 cells compared to MDA-MB-231 cells (Fig. 7). In a similar study, EpCAM aptamer (EpDT3) was used against retinoblastoma

Fig. 8. Cellular viability of the vector and vector–aptamer conjugate complexed with plasmid DNA at C/P ratio of 6 compared to PEI 25 kDa at C/P ratio of 0.8 determined in triplicate in MCF-7 and MDA-MB-231 cell cultures in 96-well trays (p value < 0.001***, P > 0.05 ns).


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Fig. 9. Analysis of the cell apoptosis by the method of PI staining in MCF-7 cells using flowcytometry. The percent of the cells in sub-G1 region, represent the percent of the apoptotic cells. (A) Untreated cells, (B) vector/siRNA C/P2, (C) vector/siRNA C/P4 trated cells, (D) vector–aptamer/siRNA-C/P2 treated cells, and (E) vector–aptamer/siRNA C/P4 treated cells.

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cancer cell lines which resulted in the internalization of the aptamer–drug conjugate into the cytoplasm and sustained drug delivery to the nucleus of cells expressing EpCAM (Subramanian et al., 2012). Another study using PEI–PEG–anti prostate specific membrane antigen (PSMA) aptamer confirmed that aptamer-mediated gene delivery could enhance transfection efficiency in LNCap cells and aptamers help to selectively destroy the cancer cells (Kim et al., 2010). Furthermore, anti-PSMA aptamer conjugated to PLGA-b-PEG nanoparticles was used to improve the tolerability and efficacy of drug therapeutic index (Dhar et al., 2008). A growing body of evidence supports the argument that malignancies originate from a small subset of cancer cells that possess unlimited proliferation potential and are capable to self-renew and to provide tumor growth. These unique populations of cells, termed cancer stem cells (CSCs), are resistant to chemotherapy and radio therapy. Studies have shown that some key signaling pathways are involved in self-renewal of stem cells

including Wnt/b-catenin, Hedgehog (Hh), and Notch signaling pathway (Subramaniam et al., 2010). Many Wnt signaling proteins act in pathways which lead to b-catenin stabilization and consequently transcriptional activation of target genes. BCL9l/ BCL9 is one the members of the nuclear complex which triggers the transcription of canonical Wnt target genes such as fgf20, dkk1, wisp1, myc, ccnd1. BCL-9l (BCL9-2) is one of the main factors regulating the switch between the adhesive and transcriptional functions of b-catenin (Adachi et al., 2004; de la Roche et al., 2008; Mani et al., 2009; Takada et al., 2012). Therefore, in the current study we evaluated the efficacy of a new SWNT–PEI–piperazine nanoparticle to transfer the BCL9l siRNA to EpCAM positive cells and investigate apoptosis induction. It was shown that PEI; which is highly efficient in DNA delivery; is less effective in siRNA applications (Zintchenko et al., 2008). In order to improve siRNA delivery efficiency of PEI-based systems, several efforts have been made. In a study a nanocomplex of PEI and mesoporous silica nanoparticles (MSNs) was designed which

Fig. 10. Analysis of the cell apoptosis by the method of PI staining in MDA-MB-231 cells using flowcytometry. The percent of the cells in sub-G1 region represent the percent of the apoptotic cells. (A) Untreated cells, (B) vector–aptamer/siRNA-C/P2 treated cells, and (C) vector–aptamer/siRNA C/P4 treated cells.


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Fig. 11. BCL9l knock down following vector–aptamer/siRNA complexes delivery to MCF-7 cells. The cells were treated with vector–aptamer/siRNA and vector–aptamer/ scrambled siRNA complexes and processed for immunoblotting with anti-BCL9l antibodies at 48 h post transfection.

Table 1 Particle size and zeta potential of SWNT–PEI–piperazine (vector)/pDNA, vector–aptamer/pDNA and PEI/pDNA complexes, at the optimum ratios for transfection activity.

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Groups

Particle size (nm)

Zeta potential (mV)

PEI C/P 0.8 Vector C/P 6 Vector–Apt C/P 6

78.1 0.9 85.0 1.9 245.3 19.9

12.6 0.1 23 3.4 44.7 1.9

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could efficiently protect and deliver siRNA into human cells (Li et al., 2013a). Another study used PEI-capped gold nanoparticles (AuNPs) for the green fluorescent protein (GFP) silencing experiment. The results of the experiment indicated that PEI-capped AuNPs induce more significant and enhanced reduction in targeted green fluorescent protein expression in MDA-MB-435s cells compared to PEI/GFP/siRNA complexes (Song et al., 2010). To test whether siRNA targeted to BCL9l would efficiently block BCL9l expression, we transfected MCF-7 cells with SWNT–PEI– piperazine/siRNA, SWNT–PEI–piperazine–aptamer/siRNA complexes at C/P ratios of 2 and 4 and compared them with that of in MDA-MB-231 cell line. These complexes should specifically bind to BCL9l mRNA and trigger its degradation. PI staining revealed that aptamer-conjugated vector/siRNA at C/P ratios of 2 and 4 induced apoptosis more efficiently than vector/siRNA in MCF7 cell line. Also the higher level of apoptosis in MCF-7 cell line induced by vector–aptamer/siRNA complex indicated that aptamer-mediated siRNA delivery could enhance transfection efficiency in EpCAM positive cells and aptamers enable us for selective destruction of cancer cells (Figs. 9 and 10). To investigate the decrease in BCL9l protein level, western blot analysis was performed. The results suggested that vector–aptamer/siRNA complex at the C/P ratio of 4 decreased the BCL9l protein level in MCF-7 cells (Fig. 11). Further study is underway to isolate breast CSCs and investigate the efficiency of the new vector used in this study to specifically transfect the CSCs cells.

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5. Conclusion

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We developed a novel EpCAM-targeted nanoparticle base on SWNT conjugated to piperazine–PEI derivative for breast cancer gene therapy. This vector–aptamer conjugate could efficiently

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increase DNA transfection. The structural modification employed in the current study also reduced cytotoxicity which enabled using polyplexes with the higher C/P ratio. Then, we successfully fabricated aptamer-conjugated nanoparticles for targeted delivery of siRNA to inhibit the expression of BCL9l in EpCAM positive cells through aptamer-mediated specific binding to the cell surface EpCAM, a well-known cancer stem cell marker. The induction of apoptosis and the decrease in BCL9l protein level indicates that this vector–aptamer/siRNA delivery system has a good potential for targeted gene delivery.

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Acknowledgements

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This work was funded by the Mashhad University of Medical Sciences, Mashhad (MUMS), Iran. Financial support from Iran Nanotechnology Initiative and Iran National Science Foundation is also acknowledged. We would like to gratefully thank Dr. Sara Amel Farzad at Pharmaceutical Research Center, MUMS and Dr. Fatemeh Kalalinia at Biotechnology Research Center, MUMS for their technical assistance. This work was included in part in the Pharm-D thesis of Marzieh Mohammadi. References Abdallah, B., Hassan, A., Benoist, C., Goula, D., Behr, J.P., Demeneix, B.A., 1996. A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum. Gene Ther. 7, 1947–1954. Abe, H., Dobson, G., Hoeger, U., Parkhouse, W.S., 1985. Role of histidine-related compounds to intracellular buffering in fish skeletal muscle. Am. J. Physiol. 249, R449–R454. Adachi, S., Jigami, T., Yasui, T., Nakano, T., Ohwada, S., Omori, Y., Sugano, S., Ohkawara, B., Shibuya, H., Nakamura, T., 2004. Role of a BCL9-related b-cateninbinding protein, B9L, in tumorigenesis induced by aberrant activation of Wnt signaling. Cancer Res. 64, 8496–8501. Akinc, A., Thomas, M., Klibanov, A.M., Langer, R., 2005. Exploring polyethyleniminemediated DNA transfection and the proton sponge hypothesis. J. Gene Med. 7, 657–663. Boussif, O., Lezoualc’h, F., Zanta, M.A., Mergny, M.D., Scherman, D., Demeneix, B., Behr, J.-P., 1995. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. 92, 7297–7301. Cai, D., Mataraza, J.M., Qin, Z.-H., Huang, Z., Huang, J., Chiles, T.C., Carnahan, D., Kempa, K., Ren, Z., 2005. Highly efficient molecular delivery into mammalian cells using carbon nanotube spearing. Nat. Methods 2, 449–454. Chen, W., Weng, S., Zhang, F., Allen, S., Li, X., Bao, L., Lam, R.H., Macoska, J.A., Merajver, S.D., Fu, J., 2012. Nanoroughened surfaces for efficient capture of circulating tumor cells without using capture antibodies. ACS Nano 7, 566–575.


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DOPE liposomes.

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Carbon Nanotubes: Solution for the Therapeutic Delivery of siRNA?

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siRNA delivery into tumor cells by cationic cholesterol derivative-based nanoparticles and liposomes.

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Progress and Challenges in Developing Aptamer-Functionalized Targeted Drug Delivery Systems.

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Aptamer-Functionalized Nanoparticles as "Smart Bombs": The Unrealized Potential for Personalized Medicine and Targeted Cancer Treatment.

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Prostate stem cell antigen antibody-conjugated multiwalled carbon nanotubes for targeted ultrasound imaging and drug delivery.