MUC1 aptamer conjugated to chitosan nanoparticles, an efficient targeted carrier designed for anticancer SN38 delivery.

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IJP 14099 1–11 International Journal of Pharmaceutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

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

MUC1 aptamer conjugated to chitosan nanoparticles, an efficient targeted carrier designed for anticancer SN38 delivery

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E. Sayari a , M. Dinarvand a , M. Amini b , M. Azhdarzadeh a , E. Mollarazi a , Z. Ghasemi a , F. Atyabi a,c, * a

Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran c Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14174, Iran b

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A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 March 2014 Received in revised form 21 May 2014 Accepted 22 May 2014 Available online xxx

Molecularly targeted therapy is of great interest for diagnosis and treatment of cancerous cells due to its low toxicity for normal cells. In this study, chitosan was utilized as a promising carrier for delivery, and aptamer (Apt) was employed for active targeting of SN38 to colon cancer. SN38 cannot be used clinically due to its poor solubility and high toxicity. Developing nanoparticles (NPs) of drug–polymer conjugates can be a good candidate for overcoming such problems. N-Carboxyethyl chitosan ester (CS-EA) was synthesized as an intermediate for conjugation of SN38 to chitosan. MUC1 DNA aptamer with 50 -NH2 functional group was conjugated to the self-assembled conjugate as a targeting agent. Prepared NPs had smooth and spherical morphology with 200 nm particle size. Conjugation of aptamer was confirmed by gel electrophoresis. In vitro cytotoxicity of NPs was assessed by HT-29 as MUC1 positive cell line through MTT assay. Aptamer conjugated NPs (Apt NPs) were more toxic than non-targeted NPs, however they were as toxic as free drug. Cellular uptake and targeting ability of prepared NPs were also confirmed via confocal microscopy. As a conclusion, prepared CS-SN38–Apt NPs can increase efficacy of drug SN38 through increasing solubility and specific delivery to the target tissue. ã 2014 Published by Elsevier B.V.

Keywords: Colon cancer Aptamer Chitosan SN38 Irinotecan Drug targeting MUC1 aptamer Nanoparticle

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1. Introduction

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Interruption of DNA synthesis in cancerous cells is an attractive method of battling cancer diseases. Topoisomerase is an essential enzyme for controlling and facilitating DNA replication by breaking and unwinding the double stranded structure of DNA, which is necessary for cell replication. Accordingly, topoisomerase could be an appropriate target for inhibition of cancerous cells propagation (Champoux, 2001). Irinotecan (CPT-11) is a semisynthetic derivative of camptothecin, exhibiting proper water solubility and hindering cell nucleic acid synthesis by inhibiting topoisomerase I enzyme function (Bala et al., 2013; Moon et al., 2008). In spite of good solubility, only 5–10% of the administrated dose will be converted to the active form of the drug (Zhang et al., 2013; Zhao et al., 2000) by human carboxylesterase (hCE). Besides, this conversion shows unpredictable inter-patient varieties. In

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* Corresponding author at: Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14117614411, Iran. Tel.: +98 21 66959052; fax: +98 21 66959052. E-mail address: [email protected] (F. Atyabi).

addition, the lacton ring of irinotecan can be easily converted to the open carboxylate form which is inactive (Gupta et al., 1997). In conclusion, these issues limit irinotecans application in cancer treatment. SN38, the active metabolite of irinotecan is 100–1000 times more potent than its prodrug, but the relatively high hydrophobicity of SN38 creates complications for its practical application (Ebrahimnejad et al., 2010). Moreover, instability of the active drug molecule at physiological pH is a major hindrance in attaining effective therapy (Bala et al., 2013). Conventional chemotherapeutic agents affect cancerous cells as well as normal cells and therefore cause various side effects. Active targeting is a potential solution which has been extensively researched in order to decrease the undesirable and fatal side effects of anticancer drugs. In active targeting, carriers such as NPs are modified with targeting agents such as antibodies or aptamers. The targeting agents will escort the drug to the desired tissue, and the amount of administered dose will be reduced; consequently, normal cells are protected from cytotoxic effects of drugs. Accordingly, preparation of appropriate NPs to enhance SN38 pharmacokinetic and efficacy and implementing targeting agents can overcome such limitations. Various macromolecular prodrugs (e.g., EZN-2208, IMMU-130)

http://dx.doi.org/10.1016/j.ijpharm.2014.05.041 0378-5173/ ã 2014 Published by Elsevier B.V.

Please cite this article in press as: Sayari, E., et al., MUC1 aptamer conjugated to chitosan nanoparticles, an efficient targeted carrier designed for anticancer SN38 delivery, Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.05.041

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(Bala et al., 2013) and nanomedicine formulations (i.e., nanoemulsions, polymeric micelles, lipid nanocapsules/nanospheres and liposomes) (Duan et al., 2010; Gu et al., 2012; Guo et al., 2012; Marier et al., 2011; Pal et al., 2005) of SN38 have been investigated to improve SN38 drug delivery to the cancer tissue, but each had advantages and weaknesses as well. Among various carriers, polymeric carriers are favorable because structural modification is relatively easy and conjugations with biocompatible polymeric carriers via covalent linkage could improve solubility and biocompatibility of the drugs. Prodrugs such as EZN-2208, which is a 40 kDa PEG conjugate (Zhao et al., 2008), PEGylated nanographene oxide (Liu et al., 2008) and HPMA_SN38 (Williams et al., 2012) are examples of conjugated drugs. Chitosan as a hydrophilic carbohydrate polymer is especially desired due to its biodegradability, biocompatibility and ability to increase drug solubility. Polymeric NPs benefit from passive targeting by enhanced permeability and retention (EPR) effect and leakage of vasculature around tumor tissue which facilitate permeation of NPs from blood vessels to the tumor tissue, besides, lack of lymphatic drainage keeps NPs at tumor site and causes accumulation of NPs at tumor tissue (Maeda et al., 2000). Implementation of active targeting in conjugation with other techniques leads to more efficient chemotherapy (Levy-Nissenbaum et al., 2008). Different kind of targeting agents have been investigated. Aptamers are oligonucleotides that fold by intramolecular interaction and acquire specific three dimensional conformation by which, they specifically bind to their antigen target with high affinity. Aptamers have attracted more attention in recent years as targeting agents, because of their many favorable characteristics such as high affinity and specificity to the target molecule, versatile selection process, ease of chemical synthesis, small physical size and lack of immunogenicity. The membrane associated glycol form of mucin glycoprotein is reported as an attractive target for anticancer drug delivery owing to its over expression in most adenocarcinomas such as colon, breast and ovarian cancers (Ferreira et al., 2009). In the current study, controlled release and targeting property were employed by using polymeric NPs which is targeted by MUC1 aptamer. As cancer cells have high expression rates of MUC1 receptor, we specifically designed chitosan NPs as a carrier for SN38 and bioconjugated MUC1 aptamer to the surface of NPs.

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2. Material and method

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2.1. Materials

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SN38 was purchased from Knowshine Pharmachemicals Inc. (Shanghai, China). Low molar mass chitosan with 90% degree of deacetylation was supplied from Primex (Karmoy, Norway). Pyridine, ethyl acrylate, sodium nitrate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were obtained from Merck (Darmstadt, Germany). Di-tert-butyl dicarbonate (BOC) (purity approximately 98%) was purchased from Sigma–Aldrich, Inc. (St. Louis, MO, USA). MUC1 aptamer (Apt) (DNA based and 50 -NH2 modification) was supplied from TAG A/S (Copenhagen, Denmark). All other chemical reagents were of analytical grade.

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2.2. Methods

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2.2.1. Depolymerization of chitosan Low molar mass chitosan was prepared as reported previously (Akhlaghi et al., 2010; Atyabi et al., 2008). Briefly, 10 mL nitrite sodium with different concentrations (2.7, 7, 14 mg/mL) was added to 100 mL of 2% (w/v) chitosan solution in

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6% (v/v) acetic acid. The reaction was continued for 1 h at room temperature while stirring. The solution pH was adjusted up to 9 by adding NaOH (5 N) drop-wise to precipitate depolymerized chitosan. The white-yellowish precipitated chitosan was filtered and washed with acetone and dissolved in acetic acid 0.1 N. Purification was carried out by subsequent dialysis against deionized water (2 1 L for 90 min and 1 1 L overnight). The product was freeze dried (LyoTrap plus LTE Scientific, UK) and stored for further studies.

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2.2.2. Synthesis of N-carboxyethyl chitosan (CS-AC) N-Carboxyethyl chitosan ethyl ester (CS-EA) was synthesized according to the method reported by Sashiwa et al. (2003a). Briefly, 250 mg of prepared chitosan was dissolved in 2% (v/v) acetic acid and then diluted with 50 mL ethanol. 0.5 mL ethyl acrylate was then added to the solution (10 equivalents/NH2), and the reaction was stirred for 48 h at 50 C temperature. The product was dialyzed against deionized water for 2 days and lyophilized to obtain CS-EA. To convert the ester of the prepared product to carboxyl group, 150 mg of lyophilized CS-EA was dissolved in 4.5 mL acetic acid (5% v/v) and kept under stirring for two days at room temperature. The reaction was ended by adding solution of NaOH (1 M) drop-wise to precipitate CS-AC. The precipitate was filtered and dried under vacuum.

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2.2.3. Synthesis of conjugated di-tert-butyl dicarbonate with SN38 (BOC–SN38) BOC–SN38 (conjugated di-tert-butyl dicarbonate with SN38) was prepared as reported by Zhao et al. (2008). Briefly, 2.45 g of SN38 was suspended in 250 mL of anhydrous dichloromethane. Furthermore, 1.764 g di-tert-butyl dicarbonate and 15.2 mL pyridine were poured into the suspension and stirred overnight at room temperature. The solution was filtered through celite, washed by HCl (0.5 N) (3 150 mL) and saturated with NaHCO3 (1 150 mL). Subsequently, the organic phase was dried over MgSO4 and then filtered and evaporated under vacuum.

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2.2.4. Conjugation of N-carboxyethyl chitosan and SN38 (CS-SN38) Conjugation of SN38 to chitosan was achieved by dissolving 100 mg of CS-AC in 5 mL PBS (pH 5.8). The reaction was continued by the addition of 200 mg EDC (5.5 molar equivalents to carboxyl group of chitosan) and was carried on for 4 h while stirring at room temperature. Afterward, 105 mg NHS (5 molar equivalents to active carboxyl group of chitosan) was added, and the reaction was stirred for 24 h at room temperature. 350 mg BOC–SN38 was dissolved in 35 mL DMSO, and the aforementioned aqueous part was added drop-wise into the solution. The reaction continued for 24 h. The reaction was ended by adjusting the pH to 8. The solution was dialyzed against methanol, deionized water (1 1 L 2:1 for 4 h and 1 1 L 1:1 for 6 h and 1 1 L 1:2 overnight) and deionized water (1 1 L for 4 h). Afterwards, the solution was lyophilized to obtain the yellowish powder that resembled chitosan. The chemical structure and reaction preparation is shown in Fig. 1.

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2.2.5. Molar mass measurement The molar mass of depolymerized chitosan was measured by Zetasizer ZS (Nano-ZS, Malvern, Worcestershire, UK) instrument using the process of static light scattering (SLS) as follows: three different concentrations of the polymer (with unknown molar mass) were prepared, and the intensity of the scattered light was measured in similar way to dynamic light scattering. Static light scattering measures the time-averaged intensity of scattered light instead of measuring the time dependent fluctuations in the scattering intensity of the molar mass and 2nd virial coefficient of the polymer can be determined. The SLS theory is based on

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Fig. 1. Schematic illustration of the procedures for the synthesis of CS-SN38 conjugate.

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Rayleigh equation, which describes the intensity of the light scattered from a particle in solution (Wyatt, 1993). KC 1 þ 2A2 C Pu ¼ Ru M where Ru is the Rayleigh ratio, M is sample molar mass, A is 2nd virial coefficient, C is concentration, Pu is angular dependence of the sample scattered intensity, and K is optical constant. 2.2.6. Conjugation confirmation To study the chemical reaction between chitosan and ethyl acrylate, the product was pressed into KBr discs and examined by Fourier transform infrared (FTIR) spectroscopy using spectrometer (FT-IR 4300 Shimadzu, Japan). 1H nuclear magnetic resonance (1H NMR) spectra was also recorded by H NMR spectroscopy (Brucker, Avanace 500 MHz, Rheinstetten Germany) to study the chemical reaction. D2O was used as a solvent. The conversion of ester to carboxyl group in chitosan modification was confirmed via FTIR spectroscopy. BOC–SN38 was dissolved in chloroform, and H NMR spectra were recorded to confirm the phenolic group protection. Finally, CS-SN38 conjugation was confirmed via H NMR spectroscopy and FTIR spectroscopy study. Thermal behavior of chitosan, CS-EA, CS-AC, SN38, CS-SN38 was also studied by differential scanning calorimetry (DSC)

(Mettler Toledi, DSC 823, Greifensee Switzerland). Briefly, the samples were put into aluminum DSC pans and sealed. Dry nitrogen was used to purge the samples at a flow rate of 10 mL/min with a temperature between 25 and 380 C and a velocity of 10 C/min.

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2.2.7. Drug content and polymer grafting Quantitation of SN38 in chitosan-SN38 conjugate (CS-SN38) was determined via spectrophotometry at 390 nm (Scnco S-3200, Seoul, Korea). In this process, different concentrations of SN38 were dissolved in methanol containing 1% trichloroacetic acid to depict the standard curve. The drug content was measured applying the following equations.

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Drug content ¼

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weight of the drug in the conjugate ð%w=wÞ weightof the conjgate

Ethyl acrylate (EA) grafting in CS-EA was measured via calculating the area under the curve (AUC) of esteric hydrogen proportional to chitosan hydrogens in NMR spectroscopic study.

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2.2.8. Preparation of NPs NPs were prepared by ionotropic gelation method. For this purpose, the CS-SN38 conjugate was dissolved in deionized water in different concentrations. Acetic acid 1% (w/w) was dropped to

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the solution, and the solution was treated with ultrasonic radiation (Misonix Ultra Sonic Liquid Processor, USA) with different programs and tripolyphosphate (TPP) (1 mL, 0.5 mg/mL) was also added during the process. 2.2.9. Nanoparticle characterization The size and zeta potential of the NPs were calculated via dynamic light scattering (DLS) (Nano-ZS, Malvern, Worcestershire, UK). All measurements were performed at a wavelength of 633 nm at 25 C with an angle detection of 90 C. Scanning electron microscopy (SEM Philips XL 30 Scanning Microscope, Philips Eindhoven, The Netherlands) was used to evaluate the morphology of NPs. For this purpose, one drop of NPs was layered on the stub and dried at room temperature. Then it was coated with gold metal using sputter coater. 2.2.10. Preparation of aptamer-modified NPs Aptamer-modified NPs were prepared as follows: NPs were suspended in 200 mL of deionized water and mixed with EDC (10 mg) and NHS (8 mg). The solution was stirred for 2 h at room temperature. Unreacted EDC and NHS were excluded by implementing Microcon1 Centrifugal Filter Device (Millipore Corporation). Consequently, 50 -NH2 aptamer was added (1% w/w) to the reacted NPs and the reaction continued for another 8 h. Finally, the unreacted aptamers were removed by centrifugation (14,000 g, 25 min). The precipitate was collected. and the acquired concentration was prepared by adding PBS (pH 7.4) (Dhar et al., 2008; Gu et al., 2009). 2.2.11. Apt–NPs conjugation confirmation Apt–NPs conjugation was confirmed by agarose gel electrophoresis using 2% (w/v) agarose in a 1 M Tris-acetate-EDTA (TAE) buffer solution. The samples were mixed with loading buffer and loaded onto the gel. Free aptamer was used as positive control. The samples were subjected to 110 V for 20 min. Then, the gel was incubated in SYBR1 SAFE solution for further 20 min and visualized under UV illumination by UV documentation device. The samples contained NPs conjugated with aptamer plus an extra aptamer (Apt–NPs)+, NPs conjugated with aptamer (Apt–NPs), native aptamer (Apt), and NPs without surface decoration. 2.2.12. In vitro cytotoxicity assay The MUC1+ human colon cancer cell line (HT-29) was obtained from national Cell Bank of Iran (Pasteur Institute, Tehran, Iran). The cells were cultured with RPMI-1640 medium supplemented with fetal bovine serum 10%, penicillin 100 units/mL and streptomycin 100 ng/mL, at 37 C in 5% CO2 humidified incubator. The cytotoxicity of CS-SN38 NPs, CS-SN38–Apt NPs and free SN38 was assessed via tetrazolium-based colorimetric (MTT) test. The tested concentrations were 20, 2.5, 0.725 mg/mL SN38 equivalents. Cytotoxicity was measured 48 after treatment. 2.2.13. In vitro cellular uptake CHO (Chinese hamster ovarian) as MUC1 cell line (Pasteur Institute, Tehran, Iran) and HT-29 cells were cultured on glass slides and treated with different NPs formulations for 2 h at 37 C. Fluorescein isothiocyanate (FITC) were loaded into NPs and NPs conjugated to aptamer as a fluorescence dye for uptake assessment. After incubation, the cells were washed with PBS three times, fixed with 4% paraformaldehyde for 20 min, and stained with DAPI for visualization of the nucleus. Finally, the cells were visualized by Nikon confocal microscope A1 (Nikon Inc., Switzerland) equipped with A1 scan head and a standard detector using a 405 nm diode laser with DAPI filter and 488 nm diode laser (Melles Griot, USA) with FITC filter.

3. Results

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3.1. Chitosan characterization

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The average molar mass of the depolymerized chitosan which were determined through SLS method are shown in Table 1. The Zetasizer Nano S and ZS measure the intensity of scattered light (K/CR) of various concentrations (C) of sample at one angle; this is compared with the scattering produced from a standard (i.e., toluene). The graphical representation of this is called a Debye plot and allows for the determination of both the absolute molar mass and 2nd virial coefficient. The Debye plot of the chitosan with desired molar mass is also shown in Fig. 2.

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3.2. Conjugation characterization

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H NMR spectroscopic study of BOC–SN38 is depicted in Fig. 3. The single peak around 1.6 ppm confirms the protection reaction. FTIR and H NMR spectroscopic study of CS-EA compared with chitosan is presented in Figs. 4 and 5. The appearance of peaks around 1.2 ppm in the H NMR spectrum and new band at 1793.3 cm1 in FTIR spectrogram of CS-EA in comparison with chitosan denotes the presence of ester group. FTIR spectroscopic study of CS-AC is also shown in Fig. 6. A peak around 1708.83 cm1 confirms the presence of carboxyl group. H NMR and FTIR spectroscopic studies of CS-SN38 are illustrated in Figs. 7 and 8. In addition to the chitosan protons, methyl, ethyl and aromatic protons of SN38 can be seen at 0.942–1.154 ppm and at 2.98–3.095 ppm and at 7.097– 8.33 ppm, respectively. The ester bond between CS-AC and SN38 can be detected from FTIR spectrum peak around 1756.58 cm1.

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3.3. Drug content and polymer grafting

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Ethyl acrylate grafting to CS-EA was 36%, and the amount of drug (SN38) conjugated to chitosan (CS-SN38) was found to be 7.09 0.12% via spectrophotometry.

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3.4. Calorimetric study of CS-SN38 conjugation

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Thermograms of SN38, chitosan, CS-AC, CS-EA, CS-SN38 and the physical mixture of SN38 and CS-EA are shown in Fig. 9. An endothermic peak can be seen around 80 C in all samples except SN38. The physical mixture thermogram, completely comply with SN38 and chitosan exothermic and endothermic peaks and totally differs from CS-SN38 conjugates thermogram.

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3.5. NPs characterization and morphology

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NPS were prepared via ionotropic gelation technique. The differently prepared formulations with various treatments are showninTable 2. Scanningelectronmicroscope imagesare presented in Fig. 10 which shows the spherical and smooth surfaced NPs.

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3.6. Electrophoresis analysis

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Conjugation of aptamer to NPs was confirmed by gel electrophoresis retardation assay which is shown in Fig. 11.

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Table 1 Different obtained molar mass of chitosan depolymerized by different concentration of NaNO2. Chitosan molar mass (kDa)

Sodium nitrite (mg/ml)

Sample

50 20 10

2.7 7 14

1 2 3


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Fig. 2. Debye plot of the desired molar mass of chitosan.

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3.7. Cytotoxicity assay

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The results of the cell viability evaluated via MTT assay is depicted in (Fig. 12). The result confirms that CS-SN38–Apt NPs have equal cytotoxicity in comparison with SN38 and increased toxicity benchmark CS-SN38 NPs mostly in higher doses.

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3.8. Cellular uptake

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The cellular uptake of the NPs and NPs conjugated with aptamer were compared on two different cell lines, HT-29 and CHO. The NPs were able to enter the cells within 1 h without causing any observed toxicity and NPs conjugated to aptamer entered HT-29 cell line more efficiently as shown in Fig. 13. The treated cells were

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examined by confocal microscopy in order to confirm that the NPs were indeed internalized by the cells and not simply bound on the surface membrane. The results indicated that the NPs decorated by aptamer were up taken more efficiently by HT-29 cells in comparison with the plain NPs. These findings indicate that the incorporation of aptamer on the surface of the NPs leads to localization of NPs in the HT-29 cells which are MUC1 positive cells. However, the observed data on the uptake study of the targeted and non-targeted NPs in CHO cell line which is MUC1 negative cell line, showed no significant difference between them. Therefore, NPs decorated with aptamer have the capability to pave the path for the safe delivery of anticancer agents to cell lines having specific receptor for aptamer while leaving healthy tissue intact.

Fig. 3. 1H NMR spectrum of BOC–SN38.


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Fig. 4. A: 1H NMR spectrum of chitosan; B: 1H NMR spectrum of N-carboxyethyl chitosan ethyl ester.

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4. Discussion

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This study aimed to investigate a targeted delivery system for SN38. CS-SN38 conjugates were synthesized via carboxylation of chitosan and esterification of the carboxyl group by conjugation with alcohol group of SN38. The chitosan depolymerization was carried out via oxidative process by NaNO2 and molar mass of the depolymerized chitosan was calculated by Zetasizer through a process called SLS. The molar mass was determined by sample measurement at different concentrations, applying the Rayleigh equation. The intensity of scattered light that a particle produces is proportional to the product of the weight average molar mass and the concentration of the particle. The Zetasizer Nano S and ZS measure the intensity of scattered light (K/CRu) of various concentrations (C) of sample at one angle; this is compared with the scattering produced from a standard (i.e., toluene). The graphical representation of this is

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called Debye plot and allows for the determination of both the absolute molar mass and 2nd virial coefficient (George and Wilson, 1994; Hiemenz, 1984). The different methods are usually used for determining the molar mass of chitosan such as employing Ubbelohde tube measuring viscosity of the medium or gel permeation chromatography (GPC) (Cherkasova et al., 2006). However, these methods are rather complicated and time consuming. In contrast, SLS is a simple and accurate method for assessing molar mass of polymers. The functionalizing of chitosan was carried out through conjugation of ethyl acrylate to depolymerized chitosan. In some studies, acrylic acid has been employed directly for the preparation of the conjugate to the chitosan (Sashiwa et al., 2003b). However, based on H NMR studies, it has been shown that ethyl acrylate is preferred for this reason as the degree of substitution is intended to increase by applying ethyl acrylate which could be affected through its higher nucleophilicity. The appearance of peaks around


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Fig. 5. A: Fourier transform infrared spectrum of chitosan; B: Fourier transform infrared spectrum of N-carboxyethyl chitosan ethyl ester.

Fig. 6. Fourier transform infrared spectrum of N-carboxyethyl chitosan.


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Fig. 7. 1H NMR spectrum of CS-SN38.

1.2 ppm in the H NMR spectrum of CS-EA indicates the presence of CH3 of ester group in addition to peaks belonging to the chitosan protons. Moreover, the appearance of new band at 1793.3 cm1 in FTIR spectrogram confirms the reaction more decisively. CS-EA was then converted to CS-AC, and this conversion was confirmed via FTIR spectroscopy. The disappearance of esteric indicator peak and appearance of carboxyl peak around 1706.83 cm1 approved this conversion. SN38 has two hydroxyl groups; one of them is phenolic and more active to enter chemical reactions. Due to the stabilization of lacton ring in SN38, 20-OH group was preferable for reaction. Therefore, SN38 phenolic group was protected via reaction with ditert-butyl dicarbonate and a single peak around 1.6 ppm in H NMR spectrum confirmed it. Ester binding of CS-AC to BOC–SN38 was carried out using EDC/NHS reagent, and conjugation was confirmed by H NMR, FTIR spectroscopic study and DSC thermogram. In addition to chitosan protons, the appearance of methyl protons from 0.942 ppm to 1.154 ppm and ethyl proton from 2.98 ppm to 3.095 ppm all owing to SN38 in HNMR spectrum confirmed the conjugation. The appearance of SN38 aromatic protons from 7.097 ppm to 8.33 ppm also confirmed the conjugation. FTIR spectrum of the conjugate confirmed the conjugation by appearance of esteric group peak in 1756.58 cm1. The deprotection of SN38 occurred during the previous step, and no other modification was needed. The DSC thermogram of the chitosan showed an endothermic peak around 80 C plus an exothermic peak around 270 C which are related to loss of water associated with hydrophilic group of polymer and decomposition of the polymer, respectively (Sakurai et al., 2000; Zeng et al., 2004). Anyhow, conjugation of ethyl acrylate to chitosan led to a shift in the peaks as shown in CS-EA thermogram while in the thermogram of SN38, the endothermic and exothermic peaks which are presented around 210 C are attributed to the melting of the molecule (Atyabi et al., 2009; Liu et al., 2014; Zhang et al., 2013). The thermal behavior of the physical mixture of these two, totally follow the SN38 and CS-EA peaks even after degradation. The absence of the peaks related to CS-EA and SN38 decomposition in CS-SN38 conjugate thermogram confirms the occurrence of the conjugation process. As mentioned above, the acylation of the 20-hydroxy group of SN38 keeps it in its closed lacton form which is critical for the antitumor activity of the drug. Moreover, the ester linkage between

Fig. 8. Fourier transform infrared spectrum of CS-SN38.


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Fig. 9. Differential scanning calorimetry of N-carboxyethyl chitosan ethyl ester, physical mixture of N-carboxyethyl chitosan ethyl ester and SN38, CS-SN38 conjugate, chitosan and SN38. 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441

chitosan and SN38 would be stable during the transport in the blood circulation increasing its stability in the blood while could easily cleaved upon the exposure to intracellular esterase which may further enhance the tumor activity of the drug (Zhang et al., 2013). The drug content of the prepared drug-conjugate measured by UV spectroscopy was about 7.1% which could be considered acceptable in comparison with some other previously reported hydrophilic prodrugs like SN38-glucuronide, IMMU-130 and EZN2208 which have a very low drug loading (3.7%). Anyhow, many of these prodrugs do not benefit from existence of the esteric bonds in the present drug-conjugate. In this study, NPs were prepared via ionotropic gelation method. NPs with long circulation time, have more possibility of exposure to the tumor tissue and could potentially accumulate at tumor site. More than 25% of nanomedicines accepted for clinical application were chemotherapy agents (Bala et al., 2013). In addition to incorporating chemotherapeutics in polymeric NPs which has more advantages in avoiding side effects and potential toxicity, many other formulations have also been investigated such as SN38-oleate a liposomal formulation (Lundberg, 1998) and NK012, a micellar drug carrier for SN38. These agents are in preclinical and clinical trials, respectively (Bala et al., 2013), but further studies are required to evaluate their effectiveness and toxicity. The size of the NPs has an enormous role in their pharmacokinetics. For instance, the hepatic uptake of the liposomes are enhanced through complement activation depending on their size. The macrophages clear larger particles, and complement system can be activated by NPs larger than 300 nm leading to their

clearance from blood (Harashima et al., 1994). In the present study, the main parameters which could affect particle size of the NPs were evaluated. It was found that the final size of the NPs depends mostly on factors such as output power and radiation time of the sonication process and the drug conjugate concentration during the preparation procedure. The smaller NPs were prepared through employing lower concentration of polymer, higher output power and higher radiation time. In contrary, the zeta potential was not affected by mentioned factors. Many studies have been carried out for active targeting of delivery systems to the target tissue. As an example, hMN-14 a hydrophilic prodrug, implemented antibody as a targeting agent. However, aptamers have many advantages over other targeting agents like antibodies. Farokhzad and Langer conjugated aptamer to cisplatin-PLGA NPs using EDC/NHS reagents for escorting the system to the prostate cancer cells (Farokhzad and Langer, 2009; Levy-Nissenbaum et al., 2008). It has been shown that the aptamer decoration will not affect the particle size of the NPs remarkably. Aptamers have small size and are interlaced among dynamic conjugate chain (Song et al., 2012). Therefore, aptamers can be considered as a more efficient candidate in comparison with other active targeting agents especially monoclonal antibodies. The gel electrophoresis was used to confirm aptamer conjugation. 4 samples were tested to verify conjugation. The wells contained native aptamer as a positive control, non-decorated NPS as negative controls because they do not stain with SYBR1 SAFE dye, NPs conjugated with aptamer plus unconjugated aptamer and conjugated NPs with aptamer in which the residual aptamer was separated via centrifugation. NPs could not move through gel and will remain at the well or will move just a little distance because of

Table 2 The changes of mean diameter of TPP-CS-SN38 nanoparticle based on different preparation conditions. Sample

Concentration (mg/ml)

Output power (W)

Sonication time (min)

Size (nm)

PDI (mV)

Zeta potential

A B C D E

2 2 2 5 10

25 50 50 50 50

5 5 2 5 5

2210.5 1750.7 3350.6 7341.2 9370.2

0.2630.052 0.2850.021 0.2350.012 0.4140.032 0.4230.012

12.50.2 11.750.12 13.20.22 14.120.3 14.350.32


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IJP 14099 1–11 10


Fig. 11. Confirmation of NPs-aptamer conjugation. A: ladder, B: NPs conjugated with aptamer plus extra aptamer (Apt NPs)+. C: NPs conjugated with aptamer (Apt NPs), D: NPs without surface decoration, E: native aptamer (Apt).

Fig. 10. Scanning electron micrograph of CS-SN38 NPs. 472 473 474 475 476 477 478 479 480 481 482 483

their size. So, stained well will be a marker for Apt NPs conjugation and band through the gel will be a marker of a free aptamer which could not be conjugated to the NPs. As it is shown in Fig. 11, sample E is native aptamer and had a band in the gel which is a marker of aptamer location. Sample D had no band in the gel and the well was not stained either, because mere NPs do not stain with SYBR1 SAFE dye. The well was stained in sample C that proves aptamer was conjugated to the NPs, but there was no band in the gel because there was no extra aptamer to move through the gel to show any band. Sample B contained residual aptamer which led to appearance of the band in the gel and the conjugated aptamer stained the well.

In vitro cytotoxicity of CS-SN38–Apt NPs, CS-SN38 NPs and free SN38 against human colorectal adenocarcinoma cell line (HT-29) was evaluated using tetrazolium based colorimetric assay (MTT) applying HT-29 cell line which is a MUC1+ cell for discriminating active targeting role of aptamer in cell cytotoxicity. In this study, in vitro cytotoxicity demonstrated diminished efficacy of CS-SN38 NPs in respect to free drug that can be related to covalent conjugation of SN38 to chitosan. Free SN38 could penetrate more efficiently and inhibit topoisomerase I by its own mechanism of action, while CS-SN38 could not access its target and consequently cytotoxic efficacy is reduced. Many other SN38 conjugates that were reported previously like PEG conjugates (EZN-2208) and hMN-14 conjugates with SN38 derivate demonstrated less toxicity in comparison with the free drug (Moon et al., 2008; Zhao et al., 2008). Although, the enhanced and selective uptake of CS-SN38– Apt by MUC1+ cancerous cells as compared with CS-SN38 nontargeted NPs compensate for reduced efficacy of conjugate. Confocal microscopy displayed enhanced and selective uptake of CS-SN8 conjugated with aptamer in comparison with nondecorated NPs on HT-29 cell line. CHO cells were also treated with CS-SN38–Apt NPs and CS-SN38 NPs, but no significant difference in cellular uptake was observed. Therefore, targeted NPs are as effective as free drug SN38. In this study targeted and nontargeted NPs overcame the insolubility issue of SN38, which limits its clinical application. Even non-targeted NPs take advantage of passive targeting and less in vitro cytotoxicity.

Fig. 12. Effect of free SN38, CS-SN38 NPs and CS-SN38–Apt NPs at different concentration on viability of HT29 cell line.


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5. Conclusion

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In this study a targeted drug delivery system based on chitosan as a carrier and DNA aptamer as a targeting agent has been developed to overcome limitations of the anticancer drug SN38, and actively deliver the drug to its site of action. We demonstrated that the targeted delivery system had the same toxicity on HT 29 cell line in respect to the free drug. Active targeting enhanced SN38 efficacy significantly because of NPs accumulation in the cancerous cells. In conclusion, by devising CS-SN38–Apt NPs, the insolubility of SN38 has been solved and by active targeting, toxic side effects of SN38 on normal cells are remarkably reduced. However, in vivo studies could further evaluate the efficacy of the present preparation more precisely.

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Acknowledgment

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The authors would like to thank the Iran National Science Foundation (INSF) for supporting this research.

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References

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