Materials Science and Engineering C 62 (2016) 771–778
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Thiolated carboxymethyl dextran as a nanocarrier for colon delivery of hSET1 antisense: In vitro stability and efﬁciency study Melika Kiani a, Farnaz Sadat Mirzazadeh Tekie a, Meshkat Dinarvand a, Masoud Soleimani b,c, Rassoul Dinarvand a,d, Fatemeh Atyabi a,d,⁎ a
Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran, Iran Stem Cell Technology Research Centre, P.O. Box 14155-3174, Tehran, Iran Department of Hematology, School of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-111, Tehran, Iran d Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 6 October 2015 Received in revised form 1 December 2015 Accepted 3 February 2016 Available online 5 February 2016 Keywords: Chitosan Thiolated dextran hSET1 antisense Colon cancer Oral delivery
a b s t r a c t Gene therapy is an optimistic approach in cancer treatment. However, for efﬁcient delivery of gene materials, designing an appropriate vector is necessary. Polyelectrolyte complexes (PECs) of chitosan and dextran could be considered a proper nanoparticulate carrier for sensitive biomaterials. In this study, PECs of chitosan and thiolated dextran were used as either an injectable or oral gene delivery system. hSET1 antisense was loaded into the PECs to suppress proliferation of colon cancer cell line. The prepared nanoparticles have ~115 nm diameter size and positive zeta potential with high mucoadhesion properties. They are able to protect antisense from degradation in serum and biorelevant ﬂuids (FaSSIF and FaSSGF). Furthermore, prepared nanoparticles demonstrated superior cellular penetration and inhibitory effect on SW480 colon cancer cell proliferation. All nanoparticles signiﬁcantly down regulated hSET1 in comparison with naked antisense. It can be concluded that thiolated PECs have potential use for injectable or oral delivery of nucleic acids such as antisense. © 2016 Elsevier B.V. All rights reserved.
1. Background In the last decades cancer is known as a genetic disease and molecular characterization of tumorigenic cells have led to identiﬁcation of several therapeutic gene targets that regulate apoptosis, proliferation and cellular signaling . Hence, gene therapy using plasmid, antisense, and siRNA became an encouraging approach in treatment of some forms of cancer such as colon cancer. Antisense oligonucleotides are small single strands of nucleic acids that are complementary to speciﬁc mRNAs in the cells . Recent clinical trials conﬁrm the ability of this class of drugs to signiﬁcantly suppress target-gene expression . Histone methyltransferases (HMTs) are key enzymes which regulate gene expression by modiﬁcation of histones . One of the essential elements of HMT complex which was ﬁrstly studied by Yadav et al. is the over expression of hSET1 in malignant cells which are responsible for histone methylation at the position of lysine 4 on histone 3 (H3K4).
⁎ Corresponding author at: Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran, Iran. E-mail addresses: [email protected]
(M. Kiani), [email protected]
(F.S. Mirzazadeh Tekie), [email protected]
(M. Dinarvand), [email protected]
(M. Soleimani), [email protected]
(R. Dinarvand), [email protected]
http://dx.doi.org/10.1016/j.msec.2016.02.009 0928-4931/© 2016 Elsevier B.V. All rights reserved.
They revealed that down regulation of hSET1 completely regresses malignant cells without affecting normal tissue. Administration of naked antisense is limited because of rapid hydrolysis by nucleases, poor cellular uptake, nonspeciﬁcity to the target cells, and low transfection efﬁciency. Therefore, development of safe and efﬁcient vector is one of the prerequisites of successful gene therapy . Many reports suggest the suitability of chitosan, a (1 → 4) copolymer of glucosamine and N-acetyl glucosamine, the only naturally occurring polycation, as a nucleic acid carrier . Chitosan offers several advantages such as biodegradability, biocompatibility, pH sensitivity, being soluble and positively charged in acidic pH . Nevertheless, minimal solubility and low buffering capacity at physiological pH lead to low transfection efﬁcacy. The presence of amine functional groups in the chitosan molecules enables it to carry a positive charge in acidic aqueous media which permits interaction with negatively charged macromolecules and to form nanoparticles by ionic cross-linking, complex coacervation, or polyelectrolyte complexation. A great variety of anionic polysaccharides can form polyelectrolyte complexes (PEC) in the colloidal size domain with chitosan, for example, carboxymethyl cellulose, alginates, carboxymethyl glucomannan, hyaluronan, heparin and dextran sulfate . This process occurs in water at room temperature without any organic solvent and with a limited energy input that makes it an appropriate approach for delivery of sensitive biomolecules. Many studies have investigated the inﬂuence of the charge-based interaction
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between chitosan and DNA on gene expression. These studies demonstrated that although increasing the molecular weight of chitosan leads to higher stability, nanoparticles do not disassemble in time for antisense to exert its biological effect [9,10]. Dextran is a family of natural polysaccharides with negatively charged groups that is widely under investigation as polymeric carriers in novel drug delivery systems . Many attempts have been made in development of dextran conjugates and their applications. The major drawback of dextran limiting its usage as a drug delivery system is its susceptibility to enzymatic digestion in human body, and high polarity which prohibits its transcellular passage . Thiolated polymers, thiomers, hydrophilic polymers modiﬁed with thiol moieties, exhibit a high transfection efﬁciency and mocuadhesive properties which make the system appropriate for oral delivery, and increases the residence time of the nanoparticles in colon. Shahnaz et al.  synthesized thiolated carboxymethyldextran (TCMD) by covalent attachment between L-cysteine and carboxymethyl dextran (CMD). Herein, we attempted to design a nanoparticulate system to take advantage of the general properties of nanomaterials in cancer therapy to increase the efﬁcacy of gene therapy. For the ﬁrst time two natural occurring polymers; chitosan and modiﬁed dextran were used as the basis of a nanocomplex system (PECs) for nucleic acid delivery via both injection and oral routs. Thiolation of dextran polymers was carried out to improve the uptake of nanoparticles and increase mucoadhesiveness which is vital for oral delivery. Since the interdisulﬁde bonds only were broken in the presence of high concentrations of glutathione in cytoplasm, such as the smart delivery system, thiolation could also ensure the preservation of the gene in serum, and release in the right place. The fabricated PECs were used for transfection of hSET1 antisense. The effects of PEC composition on stability were investigated in serum and biorelevant media. In vitro viability, cellular uptake and gene expression was studied on SW480, a colon cancer cell line. 2. Materials and methods 2.1. Materials Carboxymethyl dextran (CMD) 10–20 kDa with degree of substitution 1.3–1.5 g/mol was purchased from Sigma-Aldrich (Missouri, USA). L-Cysteine hydrochloride monohydrate, Ellman's reagent, 5,5′dithiobis-(2-nitrobenzoic acid) (DTNB), ethidium bromide (EtBr), ethylenediaminetetraacetic acid (EDTA), N-(3-Dimethylaminopropyl)N′-ethylcarbodiimide hydrochloride (EDAC), and trypsin were purchased from Sigma-Aldrich (Missouri, USA). Chitosan 400 kDa with degree of N-deacetylation 91% was acquired from Primex (Siglufjörður, Norway). Heat inactivated fetal bovine serum (FBS), Dulbecco's modiﬁed Eagle's medium (DMEM), and 3-(4, 5Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) were provided from GIBCO (Life Technologies Inc., New York, USA). All other reagents were of analytical grade from Merck (Darmstadt, Germany). Oligonucleotides antisense (5′-AAGGGGGT TCCTTGGGA-3′) and Cy5 conjugated scrambled DNA (5′-CATCGA AATCGTTGCAGTTAC-3′) were obtained from Macrogene (Seoul, Korea). Lipofectaminea 2000 was purchased from Invitrogen (California, USA).
and the reaction proceeded for additional 3 h. To purify the obtained TCMD, dialysis was performed using an 8 KDa dialysis tube (Sigma-Aldrich, Missouri, USA) against 250 ml of HCl 5 mM aqueous solution for 6 h, 500 ml of HCl 5 mM and 1% w/v NaCl aqueous solution for 12 h, 250 ml of HCl 5 mM aqueous solution for 6 h, and 500 ml HCl 1 mM aqueous solution for 12 h, subsequently. The lyophilized cysteineconjugated dextran was kept in a dark and cool place under nitrogen before use. The structure of CMD and TCMDs was afﬁrmed by Fourier transform infrared (FTIR) spectroscopy (NicoletMagna 550-FT, SpectraLab Scientiﬁc Inc., Canada). The KBr tablet of each polymer was fabricated before FTIR analysis. To determine the amount of thiol substitution on modiﬁed dextran, colorimetric reaction using Ellman's reagent, was performed. About 1.3 mg of TCMDs was hydrated in a 500 μl of 0.5 M phosphate buffer solution (PBS, pH 8.0). Then, 500 μl of DTNB 0.03% (w/v) prepared in PBS (pH 8.0) was added and the mixture was incubated 2 h at room temperature in a dark place. Absorbance of the samples was measured at 433.5 nm, and the amount of thiol was calculated using the standard curve. . 2.2.2. Chitosan fragmentation and characterization Depolymerization of chitosan was performed via nitrous acid hydrolysis according to the previously reported method . Details have been provided in supplemental materials. Average molecular weight of chitosan was estimated conducting gel permeation chromatography (GPC, Agilent Technologies, USA) using a 300 × 7.5 mm PL Aquagel-OH mixed Gel-ﬁltration column with a pore size of 8 μm, and a refractive index detector (Agilent Technologies, USA). 2.2.3. Preparation of nanoparticles containing hSET1 antisense Nanoparticles of chitosan, CMD, and TCMDs were prepared via complex coacervation. The stock solutions of CMD (500 μg/ml), TCMD (1500 μg/ml) and chitosan (9 and 18 kDa) were prepared in aqueous acetic acid solution (pH 6). Then adequate amount of antisense was mixed with above solutions. To achieve the best PEC composition, CMD, TCMDs and chitosan with different molar ratios (0.2, 1, and 5) were prepared under intense stirring (2500 rpm). The mixture was incubated at room temperature for at least 30 min. The composition of nanoparticles and component concentrations are presented in Table 1. 2.2.4. PEC characterization The surface charge of nanoparticles and average particle size were evaluated based on dynamic light scattering using Zetasizer (Malvern Instruments Ltd., UK) at 25 °C. The surface morphology of the nanoparticles was observed by scanning electron microscopy (XL 30, Philips, The Netherlands) and transmission electron microscopy (CEM 902A, Zeiss, Germany) was also used to examine the structure and topography of the nanoparticles. 2.2.5. Agarose gel retardation studies The condensation of antisense with different nanoparticles was analyzed on 2.5% w/v agarose gel containing ethidium bromide in tris–acetate–EDTA (TAE) buffer solution. Electrophoresis was carried out at 100 V for 90 min. The DNA bands were then visualized and
2.2. Methods 2.2.1. Synthesis and characterization of thiolated carboxymethyl dextran TCMD was prepared according to a previously reported method by Shahnaz et al.  with some modiﬁcation. In brief, CMD (0.5 g) was hydrated in 30 ml demineralized water. To activate the carboxylic acid groups, different amounts of EDAC (60 and 150 mM) were added, and the reaction mixture were incubated at room temperature under continuous stirring for 45 min. The pH was adjusted to 7.4 by addition of NaOH (1 M). Afterwards, 0.5 mg cysteine hydrochloride was added
Table 1 Composition of nanoparticles; nanoparticles of chitosan (9 and 18 kDa), CMD, and TCMDs prepared via coacervation, a) CMD: Carboxymethyl dextran, b) TCMD: Thiolated carboxymethyl dextran. CMDa/TCMDb to chitosan molar ratio
Chitosan (9 and 18 kDa, μg/ml)
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photographed using MultiImage™ Light Cabinet (Alpha Innotech Corporation, USA). 2.2.6. Stability of antisense loaded nanoparticles The stability of the nanoparticles in the presence of serum was investigated by agarose gel electrophoresis. 200 μl of prepared nanoparticles were incubated with 60 μl of FBS at 37 °C. DNA release was determined after 1/2, 2, 6 and 24 h using agarose gel electrophoresis described in Section 2.2.5. To investigate the stability of nanoparticles against competing polyions of the serum, 60 μl of nanoparticle containing 10 μg/ml antisenses were incubated with various amounts of heparin, 1.2, 3, and 6 μg, and stored at room temperature for 45 min followed by analyzing on 2.5% agarose gel. To mimic the conditions of the gastrointestinal track, the stability of antisense loaded nanoparticles was investigated in biorelevant media. Fasted state simulated intestinal ﬂuid (FaSSIF) and fasted state simulated gastric ﬂuid (FaSSGF) were prepared as described in Table 1 of supplemental materials. 100 μl of nanoparticles containing antisense was mixed with either FaSSGF or FaSSIF at a volume ratio of 1:1 followed by incubation at 37 °C for 30 min and 2 h, respectively. The samples were then analyzed by electrophoresis on 2.5% agarose gel. 2.2.7. Mucoadhesion study To evaluate the mucoadhesion of nanoparticles, mucin adsorption was measured by colorimetric method using periodic acid/schiff staining. 200 μl of each PEC formulation was mixed with 1 ml mucin solution (0.5 mg/ml). The samples were vortexed followed by incubation at 37 °C under continuous stirring for 1 h. The suspension was then centrifuged at 12,000 rpm for 5 min, and the supernatant was separated. 0.2 ml of periodic acid/schiff reagent was added to each supernatant followed by incubation at 37 °C for 2 h. Again, 0.2 ml periodic acid/schiff reagent was added, and after 30 min incubation at room temperature, the adsorption was quantiﬁed using spectrophotometer at 555 nm. 2.2.8. MTT assay The effect of antisense loaded nanoparticles on cancer cell proliferation was evaluated on SW480, a colon cancer cell line, using MTT assay. The cells were treated with naked hSET1, non-loaded nanoparticles, hSET1 loaded nanoparticles, and lipoplex of antisense. Lipoplex was prepared as a positive control using Lipofectamine 2000 according to the life technology™ protocol. The concentration of antisense in each well was about 5 μg/ml. After 6 h, medium was replenished with complete growth medium (pH 7.4) and cells were incubated for adequate time in the cell culture incubator following MTT assay. 2.2.9. Cellular uptake of nanoparticles The uptake of nanoparticles was investigated using laser-scanning confocal microscopy. The nanoparticles containing Cy5 conjugated oligonucleotide were prepared similar to the described method for loading hSET1 antisense. A day prior to transfection study SW480 cells were seeded in a 24-well plate to achieve 60–80% conﬂuency. Transfection was proceeded in a serum free DMEM (pH 6) by incubating the cells with nanoparticles for 2 h. Following removal of the culture medium and washing the cells twice with phosphate buffer solution (PBS, pH 7.4), SW480 cells were visualized by Nikon confocal microscope A1 (Nikon Inc., USA) equipped with A1 scan head and 32 channel A1DUS standard detector using a red diode laser (Melles Griot, USA) at 638 nm and CY5 ﬁlter. 2.2.10. Expression of hSET1 in cultured cells SW480 cells were used for knockdown experiments and the hGAPDH silencing was measured with RT-PCR. Brieﬂy, 500,000 cells per well were seeded in 6-well-plates 24 h prior to transfection and transfected with 100 pmol antisense in triplicates. Real-Time PCR
was performed using SYBR® premix Ex Taq™ Green PCR Kit and Hs_GAPDH and Hs_ACTB_2_SG Primers (Qiagen, Germany) and the Corbett RotorGene6000 manufactured and designed by Australia.
2.2.11. Statistics Statistical analysis was performed using paired t-test and one-way ANOVA with a signiﬁcance level of p b 0.05. The results are presented as a mean ± SDs.
3. Results 3.1. Synthesis and characterization of the TCMD The covalent attachment of cysteine to CMD was achieved by the formation of amide bonds between the primary amino groups of the amino acid and the carboxylic acid groups of the polymer. The product was entirely puriﬁed by dialysis against acidic deionized water. The lyophilized polymer was a white, odorless and ﬁbrous structure, and quickly soluble in water. The amounts of thiol groups per gram of polymer (μg/g) measured by Ellman's test are displayed in Table 2. Increasing the amounts of EDAC led to higher rate of thiolation. The FTIR spectra of CMD, TCMD75 (TCMD with thiol moieties of about 75 μg/g), and TCMD148 (TCMD with thiol moieties of about 148 μg/g), conﬁrmed the thiol substitution on CMD.
3.2. Chitosan depolymerization and characterization Varying the chitosan to NaNO2 molar ratio led to the production of chitosans with different molecular weights. The molecular weights of obtained chitosans were 9.12 and 17.67 kDa, similar to the intended ones, 9 and 18 kDa, respectively.
3.3. Nanoparticle preparation and characterization Nanoparticles were prepared using chitosan (9 and 18 kDa), CMD or TCMD, and hSET1 antisense by complex coacervation at acidic pH. The mean hydrodynamic diameter, polydispersity index (PDI), and zeta potentials of nanoparticles are shown in Table 3. The mean particle size varied from 96 to 260 nm. The smallest nanoparticles were achieved by TCMD-Chi 18 1:1. Using larger chitosan molecules and thiolated dextran signiﬁcantly reduced the size of nanoparticles (p b 0.05), and improved the polydispersity index (PDI) excluding nanoparticles composed of CMD which the usage of 18 kDa chitosan in their formulation caused larger particles. Altering the molecular weight of chitosan did not affect the size noticeably in nanoparticles composed of TCMD75. In spite of dextran to chitosan molar ratio which signiﬁcantly (p b 0.05) affected the surface charge of nanoparticles, increasing the chitosan Mw had no signiﬁcant inﬂuence on nanoparticles zeta potential. The SEM and TEM images of nanoparticles are illustrated in Fig. 1, respectively, which conﬁrmed the formation of nanoparticles. Table 2 Amounts of thiol groups immobilized on CMD dependent on the amount of EDAC, a) EDAC: amounts of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride used in reaction, b) TCMD75: thiolated carboxymethyl dextran with thiol moieties of 75 μg/g, c) TCMD148: thiolated carboxymethyl dextran with thiol moieties of 148 μg/g. Dextran–cysteine conjugate
Thiol groups (μmol/g)
TCMD75 c TCMD148
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Table 3 Nanoparticles characterization, a) CMD: carboxymethyl dextran, b) chi: chitosan, c) PDI: polydispersity index, d) TCMD75: thiolated carboxymethyl dextran with thiol moieties of 75 μg/g, e) TCMD148: thiolated carboxymethyl dextran with thiol moieties of 148 μg/g. Nanoparticles
Zeta potential (mV)
CMD/chi.9 CMD/chi.9 d TCMD76/chi.9 TCMD76/chi.9 e TCMD148/chi.9 TCMD148/chi.9 CMD/chi.18 CMD/chi.18 TCMD75/chi.18 TCMD75/chi.18 TCMD148/chi.18 TCMD148/chi.18
1:5 1:1 1:5 1:1 1:5 1:1 1:5 1:1 1:5 1:1 1:5 1:1
+13.9 +12.7 +13.2 +9 +18.8 +7.6 +19.4 +9.6 +20.8 +10.6 +21.2 +8.6
107 ± 30 198 ± 21 91 ± 19 158 ± 12 109 ± 10 153 ± 11 91 ± 30 115 ± 19 158 ± 28 96 ± 10 112 ± 12 98 ± 2
0.219 0.261 0.177 0.179 0.217 0.116 0.217 0.221 0.253 0.273 0.182 0.298
Fig. 2. Gel retardation assay; CMD-Chi 9 1:5(1), CMD-Chi 9 1:1(2), TCMD75-Chi 9 1:5(3), TCMD75-Chi 9 1:1(4), TCMD148-Chi 9 1:5(5), TCMD148-Chi 9 1:1(6), CMD-Chi 18 1:5(7), CMD-Chi 18 1:1(8), TCMD75-Chi 18 1:5(9), TCMD75-Chi 18 1:1(10), TCMD148-Chi 18 1:5(11), TCMD148-Chi 18 1:1(12).
3.7. Stability of nanoparticles in FBS 3.4. Gel retardation studies To examine the yield of antisense complexation with nanoparticles, agarose gel electrophoresis was performed. As shown in Fig. 2, all the nanoparticle formulations could retard the migration of antisense. However; it seems that the most complexation efﬁciency was achieved by 18 kDa chitosan and TCMD.
3.5. Nanoparticle mucoadhesion study To evaluate the mucoadhesion of the nanoparticles, samples were incubated with mucin solution. Afterwards; the amount of free mucin was measured by spectrophotometer which demonstrates the ability of nanoparticles to bind mucosal surface. Nanoparticles containing TCMD exhibited an increase in mucin adsorption as the degree of thiolation and amount of TCMD raised, indicating the enhanced mucoadhesion of TCMD (shown in Fig. 3). Mucoadhesion of nanoparticles containing 18 kDa chitosan was higher than that of 9 kDa chitosan, but it was not signiﬁcant. Also, all nanoparticles with different formulations had more mucoadhesion than the negative control.
Nanoparticles were incubated with FBS to ﬁnd out the ability of nanoparticles to protect antisense from nucleases. Since the preheated FBS was partially DNAse free, the naked antisense was almost detectable on gel (Fig. 4). After 2 h, only the CMD-Chi 18 1:5 liberated their cargo. After 6 h, most of the formulations released their antisense contents, but in the case of nanoparticles composed of TCMD148, the vague band related to the free antisense was also detected. After about 24 h, all nanoparticles dissociated and consequently the band of naked antisense was found on the gel. The fact that the intensity of the bands increased with time, indicates the ability of the formulation to release the antisense in a sustained release manner. 3.8. Stability of nanoparticles in gastrointestinal ﬂuids The stability of nanoparticles was evaluated in FaSSIF and FaSSGF. All the nanoparticles retained the antisense after 30 min incubation in FaSSIF. The nanoparticles also retained their contents in FaSSIF for 2 h. Nanoparticles of CMD-Chi 9 1:5 released the antisense in FaSSIF (Fig. 3, supplementary materials). 3.9. Cell toxicity assay
3.6. Stability of nanoparticles against polyanions Heparin was selected as a polyanionic compound to appraise the stability of antisense loaded nanoparticles against anionic components such as proteins in body ﬂuids. As illustrated in Fig. 2 (supplementary materials), the nanoparticles are absolutely stable and there was no band of released antisense attributed to the dissociation of nanoparticles.
For toxicological evaluation, nanoparticles were tested on SW480 cells and cytotoxicity was compared with lipoplex of antisense, naked antisense and antisense-free nanoparticles. The cytotoxicity was evaluated by MTT assay after 24, 48 and 72 h. The results illustrated in Fig. 5 verify that all non-loaded nanoparticles have an insigniﬁcant degree of cytotoxicity compared with control. Non-loaded nanoparticles had the lowest amount of toxicity. The toxicity level of antisense containing nanoparticles was similar to naked antisense.
Fig. 1. SEM and TEM images of the nanoparticles: SEM image of TCMD75-Chi 18 1:1 (A), SEM image of CMD-Chi 9 1:5 (B), TEM image of TCMD148-Chi 18 kDa 1:5
M. Kiani et al. / Materials Science and Engineering C 62 (2016) 771–778
Fig. 3. Mocuadhesiveness (%) of nanoparticles in comparison with control (n = 12); CMD-Chi 9 1:5(1), CMD-Chi 9 1:1(2), TCMD75-Chi 9 1:5(3), TCMD75-Chi 9 1:1(4), TCMD148-Chi 9 1:5(5), TCMD148-Chi 9 1:1(6), CMD-Chi 18 1:5(7), CMD-Chi 18 1:1(8), TCMD75-Chi 18 1:5(9), TCMD75-Chi 18 1:1(10), TCMD148-Chi 18 1:5(11), TCMD148-Chi 18 1:1(12), ethyl cellulose(13), carbomer 940.(14).
After 24 h antisense loaded CMD/9 kDa chitosan nanoparticles had higher toxicity. Nevertheless; after 48 h, the toxicity of nanoparticles containing TCMD was mounted. After 72 h the viability of SW480 cells were increased and it seems that the effect of antisense vanished.
3.10. Cellular uptake of nanoparticles The uptake of CY5-antisense loaded nanoparticles with various compositions was studied by confocal microscopy. Although all the
nanoparticles entered the cells, better uptake was observed by thiol modiﬁed samples (see Fig. 6). 3.11. Expression of hSET1 in cultured cells To investigate the gene silencing efﬁciency of various nanoparticles, SW480 cells were transfected with nanoparticles containing antisense. The transfection efﬁciency of nanoparticles was analyzed relative to GAPDH expression by real-time RT-PCR. No change in hSET expression was evident with free hSET1 antisense compared with non-transfected cells (negative controls). All the formulations effectively decreased gene expression while there was not any signiﬁcant difference between them, excluding CMD-chi 9 1:1. 4. Discussion
Fig. 4. Stability of PECs in serum after 30 min (A), 2 h (B), 6 h (C), and 24 h (D) incubation; CMD-Chi 9 1:5(1), CMD-Chi 9 1:1(2), TCMD75-Chi 9 1:5(3), TCMD75-Chi 9 1:1(4), TCMD148-Chi 9 1:5(5), TCMD148-Chi 9 1:1(6), CMD-Chi 18 1:5(7), CMD-Chi 18 1:1(8), TCMD75-Chi 18 1:5(9), TCMD75-Chi 18 1:1(10), TCMD148-Chi 18 1:5(11), TCMD148Chi 18 1:1(12).
The principal ambition of using carriers for DNA and RNA transfection is protection against degradation by nucleases. The formation of stable complexes of vector and nucleic acid is crucial for this purpose . Nanoparticles of chitosan which were reviewed speciﬁcally with dextran by Delair et al.  meet many advantages for delivery of pharmaceuticals and biomaterials including simple preparation with scalable procedure which is fast and does not require the use of chemical and hazardous solvents and reagents. Different parameters such as pH and mass ratio of CMD to chitosan noticeably affected the size and stability of complexes . Lin et al.  demonstrated that nanoparticles are autoclavable and stable for about 5 days which could be extended to 10 days by adding sugars to PEC colloidal aqueous dispersion. Moreover, recent researches presented the ability of
M. Kiani et al. / Materials Science and Engineering C 62 (2016) 771–778
Fig. 5. MTT assay of 24, 48, 72 h; the cells were treated by; CMD-Chi 9 1:5(1), CMD-Chi 9 1:1(2), TCMD75-Chi 9 1:5(3), TCMD75-Chi 9 1:1(4), TCMD148-Chi 9 1:5(5), TCMD148-Chi 9 1:1(6), CMD-Chi 18 1:5(7), CMD-Chi 18 1:1(8), TCMD75-Chi 18 1:5(9), TCMD75-Chi 18 1:1(10), TCMD148-Chi 18 1:5(11), TCMD148-Chi 18 1:1(12), naked antisense(13), lipo: antisense loaded lipofectamine(14) and control: nontreated cells(15).
these nanoparticles to enhance both humoral and cell-mediated immune response and afﬁrmed its potential as biomedical material for cancer treatment [20,21]. Thanks to advantages of chitosan and dextran nanoparticles we intended to use these nanoparticles for transfecting SW480, a colon cancer cell line, with anti-hSET1 DNA which is able to suppress the SW480 proliferation as reported by Yadav et al. . However, considering the coexistence of two negatively charged large molecules (CMD and antisense oligonucleotide), rapid dissociation of these nanoparticles in body ﬂuids especially in the presence of polyanionic components is predictable. To solve this problem and enhance the beneﬁts of nanoparticles, we intended to use thiolated dextran instead of the parental CMD. In fact, formation of disulﬁde bonds by oxidation of thiol groups along with electrostatic interactions between quaternary amine groups of chitosan, carboxylic groups of dextran, and phosphate groups of DNA causes stable nanoparticles even at lower molar ratios of chitosan to dextran.
Cysteine-conjugated CMD was prepared as dextran thiomers by amide bonds between primary amino groups of the L-cysteine and carboxylic acid groups of the polymer. The product was entirely puriﬁed by dialysis against acidic deionized water. The acidic pH was chosen at this step to minimize the formation of disulﬁde bonds . The amounts of cysteine immobilized on CMD depended on the amount of EDAC used for coupling reaction (Table 2). Increasing the amounts of EDAC led to higher rate of thiolation due to formation of more active esters participating in reaction with amines . Other parameters such as polymer and cysteine concentration can also affect the rate of cysteine substitution which was previously optimized by Shahnaz et al. . As studies showed before, complexes of high molecular weight chitosan are exceedingly stable and release their gene content slowly versus low molecular weight chitosan complexes which are easily dissociable. Although as an oral delivery system, it can better preserve the cargos and provide a higher rate of mucoadhesion . However, by postponing the release of cargos it suppressed the biological function
Fig. 6. Confocal microscopy: CMD-Chi 9 1:5 (A), TCMD75-Chi 9 1:1(B), CMD-Chi 18 1:1(C), TCMD75-Chi 18 1:1(D), TCMD75-Chi 18 1:1(E), TCMD148-Chi 9 1:5(F).
M. Kiani et al. / Materials Science and Engineering C 62 (2016) 771–778
Fig. 7. Expression of hSET1 in SW480 cells. 5 μg free hSET1 antisense or nanoparticles containing an equivalent of 5 μg of hSET1 antisense incubated with SW480 cells. CMD-Chi 9 1:5(1), CMD-Chi 9 1:1(2), TCMD75-Chi 9 1:5(3), TCMD75-Chi 9 1:1(4), TCMD148-Chi 9 1:5(5), TCMD148-Chi 9 1:1(6), CMD-Chi 18 1:1(8), TCMD75-Chi 18 1:5(9), TCMD75-Chi 18 1:1(10), TCMD148-Chi 18 1:5(11), TCMD148-Chi 18 1:1.(12).
. In this study, 9 and 18 kDa chitosan were chosen due to lower viscosity and improved solubility at physiologic pH range . As the polyelectrolyte complexation is an easy and mild method which occurs in water, this method is suitable for preparation of nanoparticles containing fragile biologic molecules that can be easily damaged during formulation . The nanoparticles were produced by vortex stirring method which was initially optimized to achieve nanoparticles with appropriate size distribution (data is not shown). It was found that molecular weight of chitosan, degree of thiolation of dextran, and chitosan: CMD molar ratio can inﬂuence particle size. Diameter size up to 150 nm is an appropriate size for cellular uptake by non-speciﬁc endocytosis; hence, aggregated large components might be excluded from cellular internalization . As shown in Table 3, all prepared nanoparticles composed of TCMD have appropriate size due to higher disulﬁde cross linking between polymer chains which prohibits swelling of nanoparticles. The chitosan–dextran nanoparticles like nano-hydrogels absorbed water when the chitosan was ionized at acidic pH. Since the test was conducted at acidic pH, the nanoparticles are in swelled shape . Raising the chitosan Mw decreased the size of nanoparticles containing TCMD148. However, this effect was not signiﬁcant in the case of CMD and TCMD75. We speculate that oxidation of thiol groups in stock solution of TCMD, especially TCMD148, caused an increase in the size of dextran molecules, and consequently 18 kDa chitosan could better condense DNA molecules. Contrary to previous studies that showed increasing dextran–chitosan molar ratio led to smaller nanoparticles , we could not ﬁnd any signiﬁcant difference between samples excluding nanoparticles of TCMD and 18 kDa chitosan. We suggest that increasing the amount of disulﬁde bonds in the case of TCMD:chitosan molar ratio 1:1 compacted the nanoparticle structure, furthermore less amounts of chitosan molecules in formulation reduced the mass of particles. The latter might be the reason for the difference between our ﬁnding and some previous reports which kept the total mass constant . Furthermore, we used polymers (CMD and chitosan) with similar Mw and quantity to efﬁciently interact with each other and neutralize some of the charges, which causes reduction in nanoparticle swelling. The lower zeta potential of these nanoparticles also conﬁrmed our hypothesis. As the number of carboxyl substitution on dextran is not 100%, but fully de-acetylated chitosan was used, the zeta potential of all nanoparticles was positive. The zeta potential of nanoparticles was reduced by escalating dextran–chitosan molar ratio. All the nanoparticles could retain the antisense during gel retardation assay, in biorelevant media, and against heparin. The formulation of nanoparticles without thiol groups showed a tendency for releasing antisense along the ﬁrst hours. Using TCMD and 18 kDa chitosan led to the formation of stable nanoparticles due to the disulﬁde cross linking and better condensation by larger chitosans. The results guarantee the thiolated nanoparticles as both oral and injectable delivery systems. Disulﬁde bonds make the nanoparticles responsive to high glutathione concentration of the intracellular media compared to lower
concentration in the extracellular ﬂuids, thus the antisense could only release into the cell and was completely protected against degradation . The results of serum stability study also conﬁrmed the higher stability of thiolated nanoparticles as naked antisense band was detected with a noticeable delay in comparison with non-thiolated ones. An increase in mucoadhesion of nanoparticles was observed when using TCMD. Using mucoadhesive polymers increases the residence times of nanoparticles in the gastrointestinal tract which provides sustained release in target tissue. The ability of thiol groups to form covalent bonds with mucin causes these effects [14,27]. Although a previous study demonstrated that larger chitosan molecules have higher mucoadhesion properties , here only negligible change in mucin adsorption was seen by altering chitosan Mw. According to results achieved by Sushma Yadav et al.  suppression of hSET1 causes the most cytotoxicity in SW480 cells. Surprisingly, we found out that naked antisense indicated signiﬁcant cytotoxicity. It might be due to the high concentration and efﬁciency of the antisense which can enter into the cell even without vector . After 24 h, antisense loaded CMD/chi 9 kDa nanoparticles had the highest toxicity. It seems that the burst release of antisense by PEC dissociation caused this phenomenon, but after 48 h the toxicity of nanoparticles containing TCMD was mounted. As it was shown in Fig. 7, CMD/Chi 9 1:5 and TCMD148/Chi 18 1:5 generated approximately 100% gene silencing which is corresponded with previous test results. Thiolation and crosslinking of TCMD caused retaining of antisense for a longer time because of more physical stability. Although it is a good feature to protect antisense against degradation, it could be a rate limiting step in gene expression. After 72 h, the viability of all nanoparticles was decreased. The temporary effect of antisense and decreasing of antisense concentration after cellular division caused this effect. As previous studies showed particle size and zeta potential strongly affects the intracellular uptake of nanoparticles. In general, smaller and more positively charged particles have higher transfection efﬁciency. In the case of colon cancer cell lines which are covered by mucin layer, thiolation can also potentially increase the uptake of nanoparticles by increasing the residence time of nanoparticles in the gastrointestinal tract . The ﬁndings of confocal microscopy were in agreement with what we expected. As shown in Fig. 7, CMD/chi 9 1:1 posed the least gene silencing efﬁcacy. We assume that endosomal escape by proton sponge mechanism and stability of these nanoparticles were less than other formulations because of lower Mw of chitosan and chitosan:CMD molecular ratio. Furthermore, the higher hydrodynamic size and lack of thiol groups decreased the uptake of nanoparticles. Surprisingly, all of the nanoparticles had almost the same effect on knock downing of hSET1. This justiﬁed the efﬁcacy of all nanoparticles during 24 h. Nevertheless, there were variations between stability and uptake of the nanoparticles, all formulations were able to transfer proper amounts of the antisense to induce adequate gene silencing.
M. Kiani et al. / Materials Science and Engineering C 62 (2016) 771–778
The efﬁcacy of nanoparticles containing TCMD148 was negligibly better which was related to increased cellular uptake. As it was mentioned before, the higher degree of thiolation led to formation of nanoparticles with appropriate size and both the thiol groups and small size guarantee cellular entry. Therefore, nanoparticles composed of TCMD148 in comparison with TCMD75 and CMD caused more efﬁcient gene silencing. 5. Conclusion Nowadays, gene therapy using plasmid, antisense, and siRNA is becoming a promising approach in treatment of various forms of cancer such as colon cancer. In this study, nanoparticles of thiolated dextran and chitosan were developed for antisense delivery which could effectively protect its contents in FBS and biorelevant media. Our study conﬁrmed the suitability of the nanoparticles as both an oral and an injectable DNA delivery system. Acknowledgment We express our sincere thanks to the Iran Stem Cell Technology Institute for the facilities provided. Authors are thankful to the ﬁnancial support from Nanotechnology Research Centre of Tehran University of Medical Sciences. The authors would like to thank Prof. Amini for providing the facility for FTIR studies and also thank N. Salamian for her kind assistance in cell culture experiments. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.02.009. References  J.E. Dancey, P.L. Bedard, N. Onetto, T.J. Hudson, The genetic basis for cancer treatment decisions, Cell 148 (3) (2012) 409–420.  M. Vanneman, G. Dranoff, Combining immunotherapy and targeted therapies in cancer treatment, Nat. Rev. Cancer 12 (4) (2012) 237–251.  S.L. Ginn, I.E. Alexander, M.L. Edelstein, M.R. Abedi, J. Wixon, Gene therapy clinical trials worldwide to 2012—an update, J. Gene Med. 15 (2) (2013) 65–77.  M. Dinarvand, M. Kiani, F. Mirzazadeh, A. Esmaeili, Z. Mirzaie, M. Soleimani, et al., Oral delivery of nanoparticles containing anticancer SN38 and hSET1 antisense for dual therapy of colon cancer, Int. J. Biol. Macromol. 78 (2015) 112–121.  S. Yadav, J. Singhal, S.S. Singhal, S. Awasthi, hSET1: a novel approach for colon cancer therapy, Biochem. Pharmacol. 77 (10) (2009) 1635–1641.  M. Dash, F. Chiellini, R. Ottenbrite, E. Chiellini, Chitosan—a versatile semi-synthetic polymer in biomedical applications, Prog. Polym. Sci. 36 (8) (2011) 981–1014.  H. Ragelle, R. Riva, G. Vandermeulen, B. Naeye, V. Pourcelle, C.S. Le Duff, et al., Chitosan nanoparticles for siRNA delivery: optimizing formulation to increase stability and efﬁciency, J. Control. Release 176 (2014) 54–63.
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