Water-Based Chitosan for Thymine Conjugation: A Simple, Efficient

Aug 22, 2016 - To avoid the use of organic solvents and/or acids, the water-based chitosan-HOBt, so-called CBt,(33) served as a good green synthesis p...
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Water-Based Chitosan for Thymine Conjugation: A Simple, Efficient, Effective, and Green Pathway to Introduce Cell Compatible Nucleic Acid Recognition Juthathip Fangkangwanwong,† Nutchanart Sae-liang,‡ Chaichontat Sriworarat,§ Amornpun Sereemaspun,*,‡ and Suwabun Chirachanchai*,† †

Department of Polymer Science, The Petroleum and Petrochemical College and ‡Nanobiomedicine Laboratory, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand § Bangkok Christian College, Bangkok 10500, Thailand S Supporting Information *

ABSTRACT: Chitosan is a potential biopolymer for cell recognition and targeting; however, when those functions are based on cationic amine groups of chitosan, cell damage is a concern. This study presents water-based chitosan conjugated with thymine (CsT) through a mild and homogeneous conjugating reaction via amide bond without the use of organic and/or acidic solvents. The CsT displays water-solubility in a wide range of pH. A series of comparative gel retardation assays confirm the selective binding with poly(A), resulting in nanoparticles of 100 to 250 nm in size. PrestoBlue cell viability assay clarifies nontoxicity and reveals noncytotoxicity to normal colon cells but inhibition of colon cancer cells. This simple pathway for water-soluble chitosan−nucleic acid leads to synergistic effects of cell compatibility and DNA recognition.

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INTRODUCTION Nucleic acid recognition is a key biological function as seen in DNA/RNA synthesis, replication, transcription, and so forth. Up to the present, various biomimic systems have been developed with the utilization of polymeric materials of which particular applications, for example, gene therapy and DNA/ RNA isolation, become feasible.1−4 In order to achieve this, molecular recognition between nucleic acid and polymeric materials under specific interactions is essential. The formation of charge−charge interaction between the phosphate anions of nucleic acid and cations of polymeric reagents such as polylysine,5−8 polyethylenimine,9,10 and chitosan11−14 is known as a practical approach. In fact, as polycations might also form a charge−charge interaction with cell membranes, the cytotoxicity leading to cell damage is a point of concern.15,16 Meanwhile, complementary base pairing under hydrogen bonds is an alternative choice, and under this interaction, cell compatibility can be expected. Based on this concept, various compounds, including peptide-sustained nucleic acids,17 Npyrrole oligomers,18 and schizophyllan,4,19,20 were reported. © 2016 American Chemical Society

However, if gene delivery is in the consideration, transfection is also required. Currently, the polymer systems, which are a combination of electrostatic interaction and hydrogen bond, allow cytotoxicity reduction and association enhancement between the polymer and nucleic acid, possibly affording transfection efficiency.21,22 At this point, the use of synthetic polymers, i.e., polyvinylimidazole21 and polyethylenimine,22 may involve questions about biodegradability when they are applied via in vivo administration and, therefore, biopolymers becomes a viable alternative. For this reason, chitosan, a naturally abundant cationic polysaccharide, is among the requirements as it is biodegradable,23,24 nontoxic,25 and tissue-compatibile,26,27 in addition to its highly positively charged amino groups. Up to now, various chitosan derivatives have been reported for nucleic acid recognition, for example, glycosylated chitosan,28 trimethylated Received: May 17, 2016 Revised: August 13, 2016 Published: August 22, 2016 2301

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Bioconjugate Chemistry chitosan,29 urocanic acid modified chitosan,30 oligoaminegrafted chitosan,31 and dendronized chitosan.32 It should be noted that as chitosan is rather inert due to its strong inter- and intramolecular hydrogen bond networks, most chemical modifications are rather complicated and require harsh conditions. At the same time, the use of various solvents has created chemical contamination, which may lead to safety issues. This study focuses on a simple, green, effective synthesis pathway to obtain a biocompatible chitosan with DNA recognition function. Furthermore, to mitigate cytotoxicity from positive charges of chitosan and induce nucleic acid recognition, thymine, one nitrogenous base, was selected as the substituent on chitosan amino groups since the base pairing between thymine and adenine can be expected. To avoid the use of organic solvents and/or acids, the water-based chitosanHOBt, so-called CBt,33 served as a good green synthesis pathway. In other words, the homogeneous CBt aqueous solution provides not only the conjugating reaction in water, but also the success of thymine conjugation in a single step, whereas the product obtained maintained its water solubility. The present work also extends the studies on binding ability with nucleic acid including cytotoxicity and anticancer and cellular uptake abilities.

Figure 1. 1H NMR spectrum of CsT in D2O.

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RESULTS AND DISCUSSION Synthesis and Characterization. This work aims to apply a water-based system and avoid the harsh conditions in conjugating thymine. Since thymine is rarely soluble in water, initially, it was modified to be a thymine-1-yl acetic acid derivative as shown in Scheme 1A (Figure SI 1a−c).

Figure 2. Solubility of CsT at different pH as identified by UV transmittance.

good water solubility at pH less than 8. The water-solubility over a wide pH range implies that the introduction of thymine groups obstructs the hydrogen bond among chitosan chains. Nucleic Acid Recognition. The ability of CsT to form complexes with nucleic acid was examined by electrophoretic mobility shift assay (EMSA) on an agarose gel. Herein, HaCaT DNA was used as a model while CsT and HaCaT DNA were mixed at various ratios. Figure 3 clarifies that the migration of DNA is completely retarded when the mass ratio of CsT to DNA was above 2.5 (lanes 3 and 4). In other words, at a ratio below 2.5, the free DNA migrates to the positive electrode (lanes 5 and 6).

Scheme 1. Synthesis of (A) Thymine-1-yl Acetic Acid and (B) CsT through CBt Aqueous Solution

The synthetic route of CsT is shown in Scheme 1B. As thymine-1-yl acetic acid and CBt are soluble in water, the preparation of CsT in a homogeneous aqueous system at room temperature utilizing EDC is favorable. 1H NMR spectrum of CsT shows chemical shifts at 1.79, 4.19, and 7.31 ppm, referring to methyl, methylene, and methine protons, respectively (Figure 1). The integral ratio between the peaks at 7.31 ppm belonging to thymine and at 2.82 ppm belonging to chitosan (at C-2) determines the degree of thymine substitution to be 50%. The transmittance of the solution in the pH range of 3 to 10 was traced by UV spectra. Figure 2 indicates that CsT shows

Figure 3. EMSA of CsT/DNA at various mass ratios. 2302

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the size of precipitated particles in the range of colloid (100− 250 nm) as a consequence of CsT/poly(A) complex formation. The study of poly(A)/CsT interaction was extended to an investigation by circular dichroism (CD). As demonstrated in Figure 6, poly(A) exhibited a positive band in the near-UV

Since thymine is a complementary base to adenine (A), the result suggests that the HaCaT DNA might be hydrogenbonded to CsT through base pairing. To confirm this, poly(A) and poly(G) oligonucleotides were applied. In this case, the binding of CsT to only poly(A) is expected. For comparative studies, CBt, which offers complexation through protonation of the amino group, is used. Figure 4 reveals that CsT binds with

Figure 6. CD spectra of (a) CsT, (b) poly(A), and (c) CsT/poly(A) mixture.

region which is characteristic of chromophores in aromatic nucleobase adenine. CsT had no ellipticity in the near-UV region. The mixture of poly(A) and CsT exhibited a total disappearance of poly(A) peak. Since the colloidal solution was formed after mixing poly(A) and CsT, the disappearance of poly(A) peak can be ascribed to the interaction of poly(A) and CsT to form colloid. It is important to note that we need to dilute our system drastically since the addition of CsT to poly(A) leads to the turbidity and it was difficult to identify the CD spectrum. Cellular Uptake. As CsT performs the poly(A) and DNA recognition, herein, the potential application of DNA or gene delivery is investigated. On this viewpoint, the cellular uptake is important information. To probe the CsT in the cell, fluorescein 5 (6)-isothiocyanate (FITC) was successfully labeled on CsT backbone (Figure SI 2). As shown in Figure 7A,B, the CsT shows the green fluorescence after the HEK293 cells are incubated with FITC-labeled CsT for 24 h and washed thoroughly with phosphate-buffered saline (PBS) solution. This indicates that the CsT was deposited in the cells. Further studies on intracellular CsT were carried out. The cells were exposed to a membrance-impermeable FITC quencher, trypan blue, prior to flow cytometric analyses. The results reveal the quenching for 6.5% trypan blue-mediated fluorescence, and this suggests cellular uptake of CsT. In Vitro Cytotoxicity. Although chitosan is known for its biodegradability, biocompatibility, and nontoxicity, uses of chitosan, in most cases, have to overcome its solubility. In fact, several reports have shown the structural modifications of chitosan to obtain solubility and DNA binding efficiency;29,30 however, its cytotoxicity as a consequence of chemical modifications is a point to be aware of. In the present work, thymine conjugation was carried out in water via a watersoluble conjugating agent (WSC). It comes to the question of whether CsT maintains chitosan biocompatibility. Therefore, colon CRL1790 cells were treated with different CsT concentration for 24 h before measuring the viable cells via PrestoBlue assay. The result reveals the cell compatibility of CsT (Figure 8). It should be noted that PrestoBlue cell viability assay is an indirect method for detecting mitochondrial metabolic activity. Therefore, the significant increase of cell viability over the control of 100% DMEM (Dulbecco’s

Figure 4. EMSA of poly(A), CsT, or CBt/poly(A) complexes, poly(G), CsT, or CBt/poly(G) complexes at various molar ratios (indicated above lane number).

poly(A) (lane 2) but not with poly(G) (lane 7). In contrast, CBt binds with both poly(A) (lane 3) and poly(G) (lane 8). This implies that the CsT/oligonucleotides complexes are formed via hydrogen bonds between the T unit in CsT and A unit in oligonucleotides, whereas CBt/oligonucleotide complexes are formed via electrostatic interaction between positively charged nitrogen in chitosan and negatively charged phosphate in oligonucleotides. However, at a low molar ratio of CsT/poly(X) or CBt/poly(X), the complexation is not identified (lanes 3, 5, 8, 10). Previously, DNA−polymer interactions through hydrogen bonding were also discussed as seen in the cases of DNA-imidazolium copolymers,21 pDNApoly(glycoamidoamine) (PGAA),22 and protein-nucleic acid.34 It is important to note that the mixing solution of CsT and poly(A) was turbid. Here, UV spectra and TEM images were traced. Figure 5A shows the decrease of UV absorbance at around 270 nm by more than double after the addition of poly(A) to CsT. This implies that poly(A) and CsT interact with each other resulting in the precipitation. Figure 5B shows

Figure 5. (A) UV spectra of (a) CsT, (b) poly(A), and (c) CsT/ poly(A) mixture, and (B) TEM image of CsT/poly(A) complexes. 2303

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the study on its anticancer activity. As shown in Figure 8, CsT allows colon CRL1790 cell growth, whereas it inhibits colon cancer SW620 cell growth. This anticancer activity implies the possibility of CsT in binding with poly(A) (a molecular target for anticancer strategies), e.g., on the 3′-termini of mRNAs of cancer cells that also present specific neo-poly(A) polymerase (PAP).36 It should be noted that the experiments were repeated more than four times to find the dose-dependent cytotoxicity except for the result at 2.5 mM. However, the reason is not clear and more investigation is under consideration. It should be noted that the results are relevant to the report by Kumar et al. in which chitosan−thymine conjugate was found to inhibit the proliferation of human liver cancer cells (HepG2) in a dose-dependent manner without cellular toxicity in noncancerous mouse embryonal fibroblast cells (NIH 3T3).37

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CONCLUSIONS This study demonstrated a simple, green, effective pathway, i.e., the mild condition in a water-based system to obtain watersoluble CsT in high yield. By simply mixing chitosan with HOBt, the CBt aqueous solution favored thymine conjugation to obtain CsT. The CsT exhibited water solubility in a wide range of pH (3 to 8). The CsT formed complexes with nucleic acid as identified from the gel electrophoresis. When CsT was allowed to form a complex with poly(A), nanoparticles ranging in size 100−250 nm were obtained. CsT allowed colon CRL1790 cell growth, while inhibiting colon cancer SW620 cell growth. At present, an extension of this work related to a potential DNA/RNA recognition reagent for an isolation system or nonviral gene delivery vector for gene and cancer therapy is in progress.

Figure 7. (A) Optical image and (B) fluorescence image of HEK293T after treating with FITC-labeled CsT.

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EXPERIMENTAL PROCEDURES

Materials. Chitosan (85% DD, MW of 3.75 × 105) was supplied from Seafresh Chitosan (Lab) Co., Ltd., Thailand. Monochloroacetic acid was purchased from Merck KgaA, The Netherlands. Thymine, fluorescein 5 (6)-isothiocyanate (FITC), sodium carbonate anhydrous, agarose, acrylamide bis-acrylamide (Acryl/BIS), tetramethylethylenediamine (TMED), ethidium bromide, SYBR green II, and Dulbecco’s modified eagle’s mediumhigh glucose (DMEM) were bought from Sigma-Aldrich, Germany. 1-Hydroxybenzotriazole monohydrate (HOBt·H2O) and 1-(dimethylamino)propyl-carbodiimide) hydrochloride (EDC·HCl) were purchased from Tokyo Chemical Industry Co., Ltd., Japan. Poly(A) (18 bases, MW of 5575.82) and poly(G) (18 bases, MW of 5863.82) were supplied from BioDesign Co., Ltd., Thailand. PrestoBlue reagent was purchased from Life Technologies, Poland. All chemicals were used as received from commercial sources without further purification. Synthesis of Thymine-1-yl Acetic Acid. Thymine-1-yl acetic acid was prepared based on a previous report with some modifications.38 Thymine (6.3 g, 0.05 mol) was dissolved in 10 wt % NaOH (100 mL). A solution of monochloroacetic acid (9.5 g, 0.1 mol) in water (25 mL) was added dropwise, while this solution was warmed to 40 °C for 12 h. The solution was cooled to room temperature and the pH was adjusted to 5.5 with conc. HCl. The solution was then allowed to stand at 0 °C for 1 h. Any precipitate (unreacted thymine) formed was removed by filtration. The solution was then adjusted to pH 2 with conc. HCl. The crystal of thymine-1-yl acetic acid was

Figure 8. Cell viability of CRL1790 and SW620 cells treated with different concentrations of CsT. Values reported are an average n = 4 ± standard deviation.

modified eagle’s mediumhigh glucose) might be due to the increase of intracellular mitochondrial dehydrogenase enzyme rather than cell proliferation. Flow cytometry was applied to affirm the cell viability. It is confirmed that the flow cytometry does not show cell proliferation (Figure SI 3). In addition, in the presence of DMSO, acetic acid, and chitosan in acetic acid, the results demonstrated the significant decrease of cell viability compared to the control, which implies cytotoxicity of those organic solvents. Since Hasegawa et al. reported the cancer cell inhibition of chitosan,35 SW620, colon cancer cells were applied to extend 2304

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(50, 10, 1, and 0.1 μL) and incubated overnight. The cells were then washed two times with phosphate buffer saline (PBS) solution before evaluation by fluorescence microscopy. To discern between intracellular and extracellular CsT, a cell impermeable FITC quencher, trypan blue, was added to the final concentration at 1 mg/mL just prior to flow cytometric analysis. Cell Viability Assay. Cytotoxicity was evaluated by using the PrestoBlue assay. The 5 × 103 cells of colon CRL1790 and colon cancer SW620 were seeded in a 96-well plate and incubated 24 h at 37 °C under 5% CO2 atmosphere in complete DMEM media supplemented with 10% fetal bovine serum. The CsT was added to the final concentrations of 5, 2.5, 1.25, and 0.625 mM. The cell viability was assessed 24 h later using the PrestoBlue Cell Viability Reagent according to the manufacturer’s instruction. The fluorescence data were read at 560 and 590 nm in a microplate reader and expressed as a percentage relative to the control cells.

achieved by cooling the thymine-1-yl acetic acid in the aqueous solution at pH 2 in a refrigerator. The crude product was collected by vacuum filtration and washed thoroughly with ethanol. The final product was dried in vacuo at 50 °C for 5 h to give 61% yield of thymine-1-yl acetic acid. 1 H NMR (500 MHz, [d6] DMSO, 25 °C, TMS): δ = 1.76 (s, 3 H; CH3), 4.36 (s, 2 H; CH2), 7.50 (s, 1 H, CH), 11.32 ppm (s, 1 H, NH); ATR-FTIR: υ bar = 1663 (CO, position 4), 1703 (CO, carboxylic acid), 1731 cm−1 (CO, position 2); MS (ESI-TOF): m/z (%) 229.0206 (100) [M+Na2−H]. Synthesis of Chitosan Conjugated with Thymine (CsT). Chitosan (1 g, 6 mmol) was stirred with HOBt (7.2 mmol, 1.2 equiv) in deionized water (100 mL) at ambient temperature until the clear solution, CBt, was obtained. Thymine-1-yl acetic acid (6 mmol, 1 equiv) was dissolved in water (200 mL) and was added into CBt aqueous solution. To the solution, EDC·HCl (18 mmol, 3 equiv) aqueous solution (100 mL) was added dropwise. The reaction was carried out at ambient temperature overnight. The crude product obtained was dialyzed and lyophilized to obtain CsT with a 98% yield. The solubility of CsT was evaluated using percent transmittance at 600 nm ranging from pH 2 to 10. 1 H NMR (500 MHz, D2O, 25 °C, TMS): δ = 1.79 (s, 3 H; thymine-CH3), 1.97 (s, 3 H; chitosan-COCH3), 2.82 (s, 1 H; chitosan-CH), 3.20−4.00 (m, 5 H; pyranose ring), 4.19 (s, 2 H; thymine-CH2), 7.31 ppm (s, 1 H, thymine-CH); ATR-FTIR: υ bar =1672 cm−1 (amide I). Gel Retardation Assay. The nucleic acid binding ability of CsT was performed by electrophoresis. HaCaT DNA was selected as the model. The CsT/DNA complexes were formulated at mass concentration ratios of 25, 2.5, 0.25, and 0.025. The mixtures were gently mixed by using the vortex for 5 s and incubated for 30 min at room temperature. The solutions were loaded onto a 2% agarose gel. Gel electrophoresis was allowed at 100 V for 30 min in 1× TAE buffer. The gels were stained with ethidium bromide and imaged using a UVilluminator. The binding efficiencies of CsT were studied using nucleic acid, poly(A), and poly(G). For the CsT/poly(A) and poly(G) complexes, the solutions were prepared at mole ratios of 100 and 10. The CBt/poly(G) complexes at the same mole ratios as CBt/poly(A) were used as the control. The mixtures were gently mixed by using a vortex mixer for 5 s and incubated for 30 min at room temperature. The complexes were loaded onto a 16% acrylamide gel containing SYBR Green II. Electrophoresis was performed at 135 V for 85 min. The gel was visualized by exposure to a UV-illuminator. Preparation of FITC-Labeled CsT. FITC-labeled CsT was prepared as reported with some modification.39 Briefly, a solution of CsT (4 mM) was prepared and the pH was adjusted to 8 by sodium carbonate buffer. FITC (1 mg/mL in DMSO) solution was added slowly into the CsT solution. After adding the required amount of FITC solution, the solution was incubated in the dark for 8 h at 4 °C. FITC-labeled CsT was precipitated in acetone and collected by centrifugation. The resulting precipitant was washed several times with fresh acetone and dried in vacuo. Cellular Uptake. HEK293 was used as a model to study the cellular uptake of CsT. The cells were seeded the night before the addition of CsT at 5 × 104 cells per well and cultured in complete DMEM media supplemented with 10% fetal bovine serum and 1% penicillin−streptomycin. FITC-labeled CsT solution (1 mg/mL) was added to the well at various volumes

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00251. FTIR, NMR, ESI-MS, and UV spectra; cell viability (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +66 81-933-7305; Fax: +66 2-215-4459 (S.C.). *E-mail: [email protected]. Phone: +66 96-569-8859 (A.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Ratchadapisek Sompote Endowment Fund (2014), Chulalongkorn University (CU-57-091-IC). J.F. gratefully thanks Rachadapisaek Sompote Fund for her postdoctoral fellowship at Chulalongkorn University. The authors acknowledge the PTT-NSTDA Chair Professor grant for the support on the present work. We thank Professor Tirayut Vilaivan at Department of Chemistry, Faculty of Science, Chulalongkorn University for the helps on circular dichroism measurement.

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ABBREVIATIONS CsT, chitosan conjugated with thymine; CBt, water-based chitosan-HOBt

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