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DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold
Journal Pre-proof DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold Pitchaya Pakornpadungsit, Thridsawan Prasopdee, Napachanok Mongkoldhumrongkul Swainson, Arkadiusz Chworos, Wirasak Smitthipong PII:
S0142-9418(19)31744-1
DOI:
https://doi.org/10.1016/j.polymertesting.2020.106333
Reference:
POTE 106333
To appear in:
Polymer Testing
Received Date: 22 September 2019 Revised Date:
21 December 2019
Accepted Date: 2 January 2020
Please cite this article as: P. Pakornpadungsit, T. Prasopdee, N.M. Swainson, A. Chworos, W. Smitthipong, DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold, Polymer Testing (2020), doi: https://doi.org/10.1016/j.polymertesting.2020.106333. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold Pitchaya Pakornpadungsita, Thridsawan Prasopdeea, Napachanok Mongkoldhumrongkul Swainsonb, Arkadiusz Chworosc, Wirasak Smitthiponga,d,* a
Specialized Center of Rubber and Polymer Materials in Agriculture and Industry (RPM), Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
b
Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
c
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90363 Lodz, Poland
d
Office of Natural Rubber Research Program, Thailand Science Research and Innovation (TSRI), Bangkok 10900, Thailand
*Correspondence to: Wirasak Smitthipong (E-mail: [email protected])
Abstract Supramolecular structure can be formed using noncovalent interactions based on the self-assembly processes. DNA is a good example for supramolecular materials because it is able to form supramolecular structure by forming specific hydrogen bonds between its base pairs. Moreover, DNA as an anionic medium can bind with oppositely charged materials to form complex structures of various shapes and properties. This work is focused on a foam complex that is formed between negatively charged DNA and positive chitosan. Various characterizations —Fourier transformed infrared spectroscopy (FTIR), Small and Wide-angle Xray scattering (SAXS and WAXS), scanning electron microscope (SEM), texture analyzer and differential scanning calorimetry (DSC) — are used to study the properties of this dried scaffold. The FTIR spectra presented the chemical structure of DNA and chitosan. While the SAXS power law decay has revealed that an increasing of chitosan content smoothens the surface of the structure, on the other hand, the roughness is much higher when the DNA content is increased. The melting point of the foam from the DSC scan has been identified. The mechanical property of foam is suitable for the application of scaffold, and there is no cytotoxicity of foam to the cell. It is expected that this type of biomaterial could be used in several applications such as functional material and as a drug delivery material. Keywords: DNA; chitosan; scaffold; biomaterials; self-assembly; supramolecular materials
Introduction Nucleic acids especially DNA is known for its biological function mainly as storage of genetic information. However, it can be also viewed as a biopolymer with nucleotides building blocks,
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connected together, form a double stranded helix. Due to its recognition potential artificial DNA molecules are being developed into new smart therapeutics. For that natural or modified DNA fragments have to be delivered inside cell, crossing hydrophobic part of the membrane. Such highly polar biopolymer is commonly encapsulated or otherwise complexed to screen negative charges. DNA can form complex structures with positively charged materials such as Lipofectamine, polyethyleneimines, positive dendrimers and natural polymers such as chitosan. Chitosan is one of the most abundant polymers found in nature. This linear polysaccharide consists of D-glucosamine and N-acetyl-D-glucosamine is formed during enzymatic deacetylation of chitin. Due to its many advantages, such as biocompatibility, biodegradability, low toxicity, biological activities, and adsorption properties chitosan is considered as efficient drug delivery carrier [1-4]. It has been applied in different forms (solutions, gels, films or fibers) [5]. DNA has been used before as a structural medium for preparation of biofilms in complex with cationic lipid, didodecyldimethylammonium bromide (DDAB) [6-8]. Results suggest that the properties of such biofilms depend on the length of DNA and the nucleic acid-lipid films have lamellar multilayered structure with layers of nucleic acids being separated by lipid bilayers of DDAB [6]. The supramolecular self-assembling films between random coil poly(styrene sulfonate) mix with DDAB (PSS-DDAB films) and double stranded DNA mix with DDAB (DNADDAB films) have been investigated [9]. It appears that DNA-DDAB film has more mechanical properties than PSS-DDAB film. So, the chain length and molecular structure of nucleic acid can also affect the mechanical and morphological properties of the films. Increase the DNA content also increases the tensile strength of the films and has more lamellar multilayer structure. The complex of DNA with chitosan was studied before, however exclusively in solution by varying the fractional content of acetylated units (FA) and degree of polymerization (DP) of chitosan [10]. The properties of DNA-chitosan polyplexes depend strongly on the FA, charge density and structure of chitosan. Increasing FA was found to increase the size and reduce the compactness of the polyplexes. The stability of polyplexes depended strongly on DP and pH, the complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. This literature work focused on stability properties of polyplexes in the aqueous environment, however, the relationship between the resulting structures and properties of DNA:chitosan material remains to be investigated. The main goal of this work was to prepare and analyze physicochemical and mechanical properties of DNA:chitosan scaffolds at different composition ratios in the dry state, calculated based on the stoichiometry of electrostatic interaction. We have generated the DNA:chitosan scaffold using the in-house developed freezedry procedure, which allow us to control the mechanical and surface properties of these scaffolds. Experimental Materials The mixture of DNA with chitosan was prepared at different molar ratios of 0.25:1, 0.5:1, 1:1, 1:0.5 and 1:0.25 DNA and chitosan respectively. Typically, a DNA sample (deoxyribonucleic acid sodium salt from salmon testes, 2000 base pairs from Sigma-Aldrich) was dissolved in 5 mL of distilled water and stirred for 30 min. The chitosan (MW ~15,000 Da from Polysciences, Inc.) was dissolved in 5 mL of 1% acetic acid and stirred for 30 min. The chitosan solution was mixed with DNA solution and stirred vigorously at room temperature for 3 h. Mixed samples were washed three times in distilled water, placed in a freezer at -20°C for 24 h followed by drying for 24 h in a freeze dryer.
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Measurements DNA:chitosan complex samples ratio of 0.25:1, 0.5:1, 1:1, 1:0.5 and 1:0.25 were tested by elemental analysis for carbon, hydrogen and nitrogen atoms (LECO CHNS-932). Structures were subjected to Small and Wide-angle X-ray scattering analysis (SAXS and WAXS) using a custom-made instrument at the x-ray diffraction facility in the Materials Research Laboratory (MRL) at UCSB, USA. The instrument utilizes a 50 µm microfocus Cu target x-ray source with a parallel beam multilayer optics and monochromator (Genix from XENOCS SA, France), high efficiency scatterless hybrid slits collimator developed in house, and Pilatus100k and Eiger 1M solid state detectors (Dectris, Switzerland). Data reduction and model fitting were carried using the NIKA and IRENA software packages developed at Argonne National Laboratory. Composition of complexes was analyzed by Fourier transform infrared spectroscopy (FTIR) using ATR-FTIR mode (BRUKER, The VERTEX 70 of the Bruker VERTEX Series) with 32 scans and wavelength range 4000-400 cm-1. Differential scanning calorimetry (DSC) (Perkin Elmer) has been employed to get thermal profile of mixed DNA:chitosan samples. All complexes were heated from 25°C to 200°C, the heating scan was performed at a scan rate of 10°C/min. Morphological structure of dried complexes was established with scanning electron microscope (SEM) model Quanta 450 FEI with tungsten filament as an electron source and typical magnification 50x. Then the micrographs from the SEM were analyzed by the ImageJ software to determine the porosity and pore size of the scaffold. Compression test was conducted by the Texture Analyzer Model TA.XTplus, using 2 mm probe with the speed of 0.03 mm/s at the depth distance of 3 mm from surface of the sample. The scaffold sample was prepared into a mold (cylindrical shape with 3 cm in diameter and height). Each of the samples was tested at least three times. Cytotoxicity of complexes was measured using standard XTT assay in aortic valve endothelial cells isolated from porcine heart valves and cultured in the laboratory. Dried complex samples of DNA:chitosan were sterilized by exposure to the UV light for 6 h. Then 1400 µL of cEGM2 (culture medium) was added into each microtube containing dry samples. A sample of conditioned media (CM-C) was used as a control. All samples CM-R1, CM-R1, CM-R2, CM-R3, CM-R4, CM-R5 corresponding to the molar ratio 0.25:1, 0.5:1, 1:1, 1:0.5 and 1:0.25, respectively, and control CM-C were incubated in the 5% CO2 at 37°C for 48 h. After the incubation all media samples were sterilized by filtering through 0.2 µm filters under the sterile condition. Porcine aortic valve endothelial cells were collected and seeded in 96-well plate at 2,500 cells per well and cultured overnight in low serum media (0.4% FBS EGM2). Then cells were incubated in conditioned CM-C and CM-R1 to CM-R5 media samples for 48 h. After 48 h incubation standard XTT assay was carried according to manufacturer’s protocol. Cell toxicity measurements were done in triplicates (n = 3). Statistical analysis was done with Kruskal-Wallis test (using ANOVA for non-parametric data). P value of P<0.05 was considered to be statistically significant. Results and Discussion The DNA:chitosan complex has been developed and studied before for targeted DNA fragments delivery into cells under the aqueous environment. Since chitosan is a natural product with biocompatibility and negligible cytotoxicity, this approach creates an interesting alternative for
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artificial polymers. Here the DNA and chitosan mixture of various ratios was obtained in the form of solid porous material in the dry state (Figure 1). This foam type of material was obtained by in-house developed freeze-dry procedure. The stoichiometry used in this study ranged from 0.25:1 to 1:0.25 for DNA and chitosan respectively, however the sample of 0.25:1 before drying looked different. As all remaining samples had semi-liquid composition otherwise known as “complex coacervation” [11], but the one with the lowest DNA content precipitated during preparation (Figure S1). In simple coacervation process, complex structure can be occurred by the electrostatic interaction between positive charge and negative charge polymers with optimum ionic strength [12, 13]. Due to chitosan is well-known as having a good complex ability [3] and has many functional groups on their back bone [1]. So, chitosan can interact with the negative charge DNA through the electrostatic interaction in order to obtain the self-assemble material. In the present study of DNA:chitosan (0.25:1) mixture, only the small content of DNA can be represented the complex precipitation. Beyond this content of DNA, the complex coacervation between higher DNA content and chitosan is obtained. To determine the theoretical charge ratio of DNA to chitosan we estimated for DNA to be 325g/base and therefore per charge and for chitosan to be 161g/unit. With this estimation the theoretical ratio for 1:1 charge would be in the sample of 0.5:1 for DNA:chitosan mixture. For other samples the remaining charges in the mixture are screened by residual counterions. After that, all the complex samples were washed three times in distilled water, placed in a freezer at -20°C for 24 h followed by drying for 24 h in a freeze dryer. Finally, the DNA:chitosan scaffolds are obtained (Figure 1). All scaffold samples were first tested for its composition using elemental analysis, Fourier transformed infrared spectroscopy and glass transition melting temperature. Elemental analysis of carbon, hydrogen and nitrogen atoms in the DNA:chitosan dried complex for selected samples was compared with theoretical values (Table 1).
Figure 1 Picture of scaffold foam type material obtained from the mixture of DNA with chitosan in the ratio of 0.25:1, 0.5:1, 1:1, 1:0.5 and 1:0.25 respectively.
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Some discrepancy in results can be associated with the DNA composition, which consists of four types of nucleobases with different element contents. Differences might be also due to the remaining counter-ions mainly from the chitosan, as DNA was used in the sodium salt form. Nonetheless the composition of hydrogen and nitrogen seem to be correct. DNA has four types of base pairs and each base pair has different element contents. Table 1 Elemental analysis of DNA:chitosan complexes in ratios 0.5:1, 1:1 and 1:0.5 (C: carbon atom, H: hydrogen atom and N: nitrogen atom). DNA:chitosan ratio
a
Theoretical result (wt%) a
Experimental result (± 1 wt%)
C
H
N
C
H
N
0.5:1
35
5
7
33
6
9
1:1
39
5
11
31
5
14
1:0.5
38
5
12
30
5
13
Theoretical elemental of DNA:chitosan by calculation.
Fourier transformed infrared spectroscopy spectra were collected for pure samples of DNA and chitosan (Figure S2, S3) and results are in good agreement with those present in the literature [5, 13]. When DNA was mixed with chitosan the FTIR spectral analysis shows characteristics for both species (Figure 2). Bands at 3353, 3218 and 2876 cm-1 are associated with the -OH stretching vibration of pyranose ring, the NH stretching vibration in primary amide and to the CH asymmetric stretching vibration in the chitosan ring, respectively. Bands at 1651, 1586 and 1376 cm-1 are characteristic for N-acetyl groups with the C=O, stretching vibration in primary amide, the NH bending in secondary amide and CN stretching vibration in tertiary amide, respectively. The absorption band at 1416 cm-1 corresponds to amino groups. The symmetrical deformation for CH3 can be observed at 1316 cm-1. The vibration C-O-C glycosidic linkage is observed at 1151 cm-1. The sugar phosphate backbone vibration range with the asymmetric stretching of phosphate groups can be found at 1220 cm-1, the symmetric stretching of phosphate groups at 1060 cm-1 and C-C stretching vibration (deoxyribose) at 964 cm-1. The signal at 1018 cm-1 suggests that DNA adopts B type conformation [7]. It can be noticed that multiple characteristic peaks in FTIR spectra for DNA are partially separated from peaks for chitosan, thus it is easy to ascertain that the mixture composes both molecules, however, it is impossible to correlate the signal with the composition based on this analysis. Differential scanning calorimetry (DSC) was used to monitor heat effects associated with phase transitions and physical properties of scaffold as a function of temperature and also establish melting temperatures for all DNA:chitosan mixture complexes (Figure S4). Distinctive endothermic peak in the melting temperature (Tm) for dried complexes can be found in 5 mixtures (from 0.25:1 to 1:0.25). DSC values collected in Table 2 show gradual increase of the melting temperature with the increase of the DNA content. This might be due to the approximate size of DNA molecules as compared to chitosan (DNA molecules for 2,000 base pairs is approximately 1,300 kDa and chitosan is 15 kDa). Also, the double helix DNA structure might contribute to the thermal stability of the complex. This result is similar to the previous studies, a board endothermic peak, for solid DNA and DNA in solution [14-17], is observed which corresponds to DNA melting.
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1219-962 cm -1 1710-1484 cm -1
A 1416-1376 cm -1
B
Absorbance
C
D
E
F
G 3500 3300 3100 2900 2700
Wavenumber (cm-1)
1800
1600
1400
1200
1000
800
Wavenumber (cm-1)
Figure 2 The FTIR spectra of (A) DNA alone, (B) chitosan alone, (C) DNA:chitosan = 0.25:1, (D) DNA:chitosan = 0.5:1, (E) DNA:chitosan = 1:1, (F) DNA:chitosan=1:0.5 and (G) DNA:chitosan = 1:0.25. Table 2 Melting point and glass transition temperature in the DNA:chitosan ratios from 0.25:1, to 1:0.25. DNA:chitosan ratio
Melting temperature (± 1°C)
0.25:1 0.5:1 1:1 1:0.5 1:0.25
60 70 73 72 72
Once the composition of the DNA:chitosan complex was established and melting temperature profile suggests that homogeneous mixture is formed, we were curious how the structure looks like and whether it is influenced by ratio change. For structural analysis the Small and Wide-angle X-ray scattering (SAXS and WAXS) and scanning electron microscopy (SEM) were used. The length scale range for SAXS and WAXS analysis was 0.1 nm - 100 nm (Figure 3). The SAXS region corresponds to the larger scale structures (up to 100 nm), and the
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main characteristics here can be described by Porod scattering. In the general power-law decay, linear Log(I) vs Log(Q) the intensity can be explained as 1/QP, where P is the Porod parameter or power law exponent. This parameter correlates to the geometry and interface roughness of the scattering objects and thus can be used to differentiate between different structures. The results show some differences in the SAXS analysis of different DNA:chitosan ratios. The dried complexes in ratios of 0.25:1, 0.5:1 and 1:1 show similar scattering behaviour, with a power law decay ~1/Q4 suggesting smooth surface of samples. However, dried complexes in the ratio 1:0.5 and 1:0.25 show scattering behaviour, with the power law decay ~1/Q3, suggesting the rough surface structure. Essentially when the chitosan content is high the surface is rather smooth and when DNA content is increased the roughness much higher. High Porod parameters are usually assigned to the large 3-dimensional structures (>100 nm). In other words, what we are seeing is not the size of the object, but rather the surfaces within. Additionally, there are no obvious diffraction peaks present in the data, which indicates that there is no ordering at the large length scale which SAXS probes [18-20]. Concurrently the WAXS experiment shown different profile depending on the DNA:chitosan ratio. Individually both, DNA and chitosan have been investigated by X-ray diffraction previously [21, 22]. In the DNA:chitosan complexes, with ratio close to 1:1 we can recognize two groups of signals; at Q ≈ 1.85 A-1 for DNA and at Q ≈ 0.75 A-1 for chitosan. The DNA:chitosan complexes at ratios 0.25:1 and 1:0.25 present broad peaks with no distinctive characteristics [18-20]. However, beyond these basic descriptions of the data, we do not have a good solid model to explain the features because the scaffold structure could be quite complex. In anyway, Tirrell and co-workers have successfully prepared the supramolecular self-standing film based to electrostatic interaction between anionic DNA and cationic amphiphile (DDAB) [4]. They found that the SAXS data for DNA-DDAB sample shows similar harmonic peaks, suggesting a lamellar-like structure with layers of DNA being separated by lipid bilayers of DDAB. This result is in agreement with the results obtained previously by Safinya [10]. However Tirrell’s samples were dried samples, whereas Safinya’s samples were analyses in the aqueous solution. In the previous work, we investigated the supramolecular film between 100 nucleotides long poly(adenylic acid) and 15-30 nucleotides short RNA [4]. We found that the film’s morphology changes (analyzed by AFM), height images show a rougher surface when we increase the concentration of long poly(adenylic acid) in the mixture and plateau at around 25 wt% of poly(A).
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Figure 3 SAXS and WAXS analysis of DNA:chitosan dried samples in five ratios, from 0.25:1 to 1:0.25. SAXS data (upper Figure) represents short scale of Q [A-1] whereas WAXS data (lower Figure) represents longer scale of Q [A-1]. In the SAXS region, the Porod model of general power-law decay (1/QP where P is called the Porod parameter) can be applied. The Porod parameter of 4 and 3 can be interpreted as the dried complexes structure had a smooth and rough surface structure, respectively. The Porod parameter of 3 was better order then the Porod parameter of 4. In the WAXS region (lower Figure), there are two groups of peak which are from DNA (Q ≈ 1.85 A-1) and chitosan (Q ≈ 0.75 A-1) separately. Morphological characteristics were acquired by SEM (Figure 4). From the first glance we can see that all dried complexes have a porous-type structure. This is very promising in the view that these dried complexes might be used as a scaffold. Pore size is a very important factor for mechanical, elastic, cell adhesion, migration and proliferation properties. It can be divided into nano-size (nano-roughness, <100 nm), micro-size (micro-roughness, 100 nm - 100 µm) and macro-size (100 µm – 1 mm). The porosity for obtained structures was quantified based on the ImageJ contrast analysis (Figure 4 right).
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Figure 4 SEM analysis (magnification 50x) of DNA:chitosan samples in five ratios, from 0.25:1 to 1:0.25 from A to E respectively: (left panel) presents examples of SEM images as collected during analysis, (right panel) presents the two-color scale contrast as it was used for porosity analysis by ImageJ software.
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Table 3 Pore size and percent porosity for five dried DNA:chitosan complex samples analyzed by ImageJ software. Porosity (± 1 %) Pore size (± 1 µm) DNA:chitosan ratio 0.25:1 0.5:1 1:1 1:0.5 1:0.25
105 – 1294 121 – 650 75 – 447 98 – 845 144 – 937
62 60 58 51 49
The results of pore size and percent of porosity for all 5 dried samples shown large differences in the pore size, ranging from macron sizes, 100 µm up to 1 mm. In the scaffold to be considered as biomaterial for cell growth or bio-mineralization, the macro-pore size plays an important role. For instance, cell seeding, cell distribution, migration, differentiating and further neo-vascularization in vivo is dependent on the material’s surface properties and also on the porosity [23]. The porosity is measured in the black areas compared to the total areas (Figure 4) using the ImageJ software, it was found that porosity of scaffold tends to decrease with increasing of DNA content. This could be explained by the fact that DNA molecule is bigger than chitosan molecule (DNA molecule is 2000 base pairs or ≈ 1300 kDa while chitosan molecule is 15 kDa). Moreover, the structure of DNA molecule is a double helical structure while the structure of chitosan molecule is a linear polysaccharide. So, DNA molecule can affect the porosity of the dried complexes. The highest homogeneity in pore size has a 1:1 complex, however those from a ratio of 0.5:1 to 1:0.5 can be considered for cell growing, generating and differentiating the cells into organs or cardiovascular supportive structure. Although, we observed small differences in the SEM images, these probably would not count for the differences between low and high DNA content in samples observed in SAXS. These are most probably manifestation of fine nanostructure organization. We also studied the mechanical properties of the scaffolds for further use in materials applications such as tissue engineering, etc. Compressive strength values for each type of scaffold were determined at least in triplicate. Statistics on a completely randomized design were determined using Statgraphic software (release 5.0, Statgraphic Corp.) to calculate analysis of variance (ANOVA). Bonferroni’s multiple range test was used to determine significantly different averages at a 95% confidence interval [24]. We found that the compressive strength of scaffolds increases with increasing of DNA content (Table 4). This result is in good agreement with the melting point and morphological properties of the scaffolds, DNA molecule could also affect the mechanical properties of this type of scaffold. So, we can design and develop a tailor-made scaffold for the specific application, in particular the DNA:chitosan scaffold with optimum mechanical properties. Regarding to a soft material as foam rubber, its compressive strength can be varied during 0.01-30 MPa depending on the foam formulation and density [25, 26]. Generally, scaffold should provide a mechanical strength in the range of 0.05-350 MPa. For example, the moduli of poly(ethylene glycol)-terephthalate scaffolds were 0.05-2.5 MPa in dry state [27]. Table 4 Compressive strength for five dried DNA:chitosan scaffold ratios from 0.25:1 to 1:0.25. DNA:chitosan ratio
Compressive strength (± 0.01 MPa)
0.25:1 0.5:1
0.20 0.35
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DNA:chitosan ratio
Compressive strength (± 0.01 MPa)
1:1 1:0.5 1:0.25
0.56 0.54 0.52
In anticipation of this material to be used as a biocompatible platform to grow cell and tissue we want to investigate the cytotoxicity and cell compatibility. The most important is whether prepared complexes will release toxic material into medium. For that DNA:chitosan samples were treated with cell growth media and then XTT cytotoxicity test was performed after 48 h incubation. We used porcine aortic valve endothelial cells (PAVEC) as a model cellular system. Results shows no cytotoxicity, actually one can notice slight increase in proliferation after incubation with DNA:chitosan complex (Figure 5). The complex composition has also very small effect on cell viability, with the statistical threshold setup at P<0.05 there is no statistical difference between CM-R1 to CM-R5 samples.
Figure 5 Cell cytotoxicity measurement using XTT test after 48 h porcine aortic valve endothelial cells treatment with DNA:chitosan complex samples CM-R1 to CM-R5 of 0.25:1 to 1:0.25 DNA to chitosan respectively. Conclusions We demonstrate efficient method of coacervation complex preparation using anionic DNA and cationic chitosan in ratios ranging from 0.25:1 to 1:0.25 (DNA to chitosan). During desiccation all samples form foam scaffold type of structure. The composition of dried samples was confirmed using elemental analysis, Fourier transformed infrared spectroscopy and differential scanning calorimetry. The structure of newly created scaffold was analyzed with Small and Wide-angle X-ray scattering (SAXS and WAXS) and scanning electron microscopy (SEM). Surprising to us was that when the chitosan content is high (ratio 0.25:1, 0.5:1 and 1:1) the surface is rather smooth and when DNA content is increased (ratio 1:0.5 and 1:0.25) the roughness is much higher, as suggested by SAXS power law decay. As SEM analysis indicates, structures of all dried complexes have relatively large-scale pores (100 - 1000 µm), however, percentage porosity decrease with increase of DNA content in the sample. The mechanical property of foam is suitable for the application of scaffold. Very interesting for us was to find out that all samples have no effect on cell toxicity and proliferation. This was tested using primary porcine aortic valve endothelial cells, it would be interesting to see similar results on other cell lines especially those where contact with implants and artificial scaffold is anticipated. In
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perspective, these types of material could be useful for medical application as a functional material, and drug delivery system. Acknowledgements The authors gratefully acknowledge the financial support of the Kasetsart University Research and Development Institute (KURDI), Bangkok, Thailand. Moreover, the authors particularly grateful for the assistance given by Dr. Youli Li and Mr. Phillip Kohl for SAXS and WAXS measurements from Materials Research Laboratory (MRL), University of California, Santa Barbara, USA. The SAXS and WAXS research carried out here made extensive use of shared experimental facilities of the MRL: an NSF MRSEC, supported by NSF DMR 1720256. The MRL is a member of the NSF-supported Materials Research Facilities Network (www.mrfn.org). Data availability The raw/processed data required to reproduce these findings cannot be shared at this time due to legal or ethical reasons.
References and notes [1] S. Kim, M.E. Nimni, Z. Yang, B. Han, Chitosan/gelatin–based films crosslinked by proanthocyanidin, Journal of Biomedical Materials Research Part B: Applied Biomaterials 75B(2) (2005) 442-450. [2] R. Jayakumar, N. Nwe, S. Tokura, H. Tamura, Sulfated chitin and chitosan as novel biomaterials, International Journal of Biological Macromolecules 40(3) (2007) 175-181. [3] M.M.F.A. Baig, M. Naveed, M. Abbas, W. Chunxia, S. Ullah, M. Hasnat, A. Shad, M. Sohail, G.J. Khan, M.T. Ansari, DNA scaffold nanoparticles coated with HPMC/EC for oral delivery, Int J Pharm 562 (2019) 321-332. [4] P. Pakornpadungsit, W. Smitthipong, A. Chworos, Self-assembly nucleic acid-based biopolymers: learn from the nature, Journal of Polymer Research 25(2) (2018) 45. [5] M. Rinaudo, Chitin and chitosan: Properties and applications, Progress in Polymer Science 31(7) (2006) 603-632. [6] W. Smitthipong, T. Neumann, A. Chworos, L. Jaeger, M. Tirrell, Supramolecular Materials Comprising Nucleic Acid Biopolymers, Macromol. Symp. 264 (2008) 13-17. [7] R. Chollakup, W. Smitthipong, Chemical structure, thermal and mechanical properties of poly(nucleotide)–cationic amphiphile films, Polymer Chemistry 3(9) (2012) 2350-2354. [8] S. Yasmeen, M. Kabiraz, B. Saha, M. Qadir, M. Gafur, S. Masum, Chromium (VI) Ions Removal from Tannery Effluent using Chitosan-Microcrystalline Cellulose Composite as Adsorbent, 2016. [9] R. Chollakup, W. Smitthipong, A. Chworos, Supramolecular cooperative-assembly of polyelectrolyte films, RSC Advances 3(14) (2013) 4745-4749. [10] S.P. Strand, S. Danielsen, B.E. Christensen, K.M. Vårum, Influence of Chitosan Structure on the Formation and Stability of DNA−Chitosan Polyelectrolyte Complexes, Biomacromolecules 6(6) (2005) 3357-3366. [11] R. Chollakup, W. Smitthipong, C.D. Eisenbach, M. Tirrell, Phase Behavior and Coacervation of Aqueous Poly(acrylic acid)−Poly(allylamine) Solutions, Macromolecules 43(5) (2010) 2518-2528.
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[12] H. Bohidar, Coacervates : A novel state of soft matter - An overview, J. Surface Sci. Technol 24 (2008) 105-124. [13] A.S. Hoffman, Hydrogels for biomedical applications, Advanced Drug Delivery Reviews 64 (2012) 18-23. [14] J.G. Duguid, V.A. Bloomfield, J.M. Benevides, G.J. Thomas, Jr., DNA melting investigated by differential scanning calorimetry and Raman spectroscopy, Biophys J 71(6) (1996) 33503360. [15] Y.S. Tarahovsky, V.A. Rakhmanova, R.M. Epand, R.C. MacDonald, High temperature stabilization of DNA in complexes with cationic lipids, Biophys J 82(1 Pt 1) (2002) 264-273. [16] S.L. Lee, P.G. Debenedetti, J.R. Errington, B.A. Pethica, D.J. Moore, A Calorimetric and Spectroscopic Study of DNA at Low Hydration, The Journal of Physical Chemistry B 108(9) (2004) 3098-3106. [17] Y. Liu, F. Tan, Calorimetric studies of thermal denaturation of DNA and tRNAs, Journal of thermal analysis 45(1) (1995) 35-38. [18] Y. Li, R. Beck, T. Huang, M.C. Choi, M. Divinagracia, Scatterless hybrid metal-single-crystal slit for small-angle X-ray scattering and high-resolution X-ray diffraction, Journal of Applied Crystallography 41(6) (2008) 1134-1139. [19] J. Ilavsky, P.R. Jemian, Irena: tool suite for modeling and analysis of small-angle scattering, Journal of Applied Crystallography 42(2) (2009) 347-353. [20] J. Ilavsky, Nika: software for two-dimensional data reduction, Journal of Applied Crystallography 45(2) (2012) 324-328. [21] C.R. Safinya, Structures of lipid–DNA complexes: supramolecular assembly and gene delivery, Current Opinion in Structural Biology 11(4) (2001) 440-448. [22] G.L. Clark, A.F. Smith, X-ray Diffraction Studies of Chitin, Chitosan, and Derivatives, The Journal of Physical Chemistry 40(7) (1935) 863-879. [23] I. Bružauskaitė, D. Bironaitė, E. Bagdonas, E. Bernotienė, Scaffolds and cells for tissue regeneration: different scaffold pore sizes—different cell effects, Cytotechnology 68(3) (2016) 355-369. [24] R. Chollakup, J. Suesat, S. Ujjin, Effect of Blending Factors on Eri Silk and Cotton Blended Yarn and Fabric Characteristics, Macromolecular Symposia 264 (2008) 44-49. [25] R. Suksup, Y. Sun, U. Sukatta, W. Smitthipong, Foam rubber from centrifuged and creamed latex, Journal of Polymer Engineering 39 (2019). [26] Y. Zhu, G. Luo, R. Zhang, Q. Liu, Y. Sun, J. Zhang, Q. Shen, L. Zhang, Investigation of the Constitutive Model of W/PMMA Composite Microcellular Foams, Polymers 11(7) (2019). [27] V. Vishwanath, K. Pramanik, A. Biswas, Optimization and evaluation of silk fibroin-chitosan freeze-dried porous scaffolds for cartilage tissue engineering application, Journal of Biomaterials Science, Polymer Edition 27(7) (2016) 657-674.
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DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold Pitchaya Pakornpadungsita, Thridsawan Prasopdeea, Napachanok Mongkoldhumrongkul Swainsonb, Arkadiusz Chworosc, Wirasak Smitthiponga,d a
Specialized Center of Rubber and Polymer Materials in Agriculture and Industry (RPM), Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand c Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90363 Lodz, Poland d Office of Natural Rubber Research Program, Thailand Science Research and Innovation (TSRI), Bangkok 10900, Thailand Correspondence to: Wirasak Smitthipong (E-mail: [email protected]) Highlights: • • • • •
Porous scaffold can be obtained by self-assemble complex between DNA and chitosan. Composition of dried scaffolds was confirmed by elemental analysis, FTIR and DSC. Structure of newly created scaffold was analyzed by SAXS & WAXS and SEM. Mechanical property of foam is suitable for the application of scaffold. Scaffold samples have no effect on cell toxicity and proliferation.
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DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold Pitchaya Pakornpadungsita, Thridsawan Prasopdeea, Napachanok Mongkoldhumrongkul Swainsonb, Arkadiusz Chworosc, Wirasak Smitthiponga,d a
Specialized Center of Rubber and Polymer Materials in Agriculture and Industry (RPM), Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand c Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90363 Lodz, Poland d Office of Natural Rubber Research Program, Thailand Science Research and Innovation (TSRI), Bangkok 10900, Thailand Correspondence to: Wirasak Smitthipong (E-mail: [email protected]) Conflicts of Interest: The authors declare no conflict of interest in reported research. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
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DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold Pitchaya Pakornpadungsita, Thridsawan Prasopdeea, Napachanok Mongkoldhumrongkul Swainsonb, Arkadiusz Chworosc, Wirasak Smitthiponga,d a
Specialized Center of Rubber and Polymer Materials in Agriculture and Industry (RPM), Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand c Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90363 Lodz, Poland d Office of Natural Rubber Research Program, Thailand Science Research and Innovation (TSRI), Bangkok 10900, Thailand Correspondence to: Wirasak Smitthipong (E-mail: [email protected]) Author statement: P.P. carried out the preparation and characterization of scaffold; T.P. carried out the SAXS and WAXS; N.M.S. carried out the cytotoxicity assay; A.C. contributed to the discussion and review of the manuscript; W.S. conceived the study, designed the study and helped with the manuscript. This manuscript and its contents, has not been published previously and is not under any consideration for publication in any other journal at the time of this submission. All authors gave final approval for publication and agree to be held accountable for the work performed therein.
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S0142-9418(19)31744-1
DOI:
https://doi.org/10.1016/j.polymertesting.2020.106333
Reference:
POTE 106333
To appear in:
Polymer Testing
Received Date: 22 September 2019 Revised Date:
21 December 2019
Accepted Date: 2 January 2020
Please cite this article as: P. Pakornpadungsit, T. Prasopdee, N.M. Swainson, A. Chworos, W. Smitthipong, DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold, Polymer Testing (2020), doi: https://doi.org/10.1016/j.polymertesting.2020.106333. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold Pitchaya Pakornpadungsita, Thridsawan Prasopdeea, Napachanok Mongkoldhumrongkul Swainsonb, Arkadiusz Chworosc, Wirasak Smitthiponga,d,* a
Specialized Center of Rubber and Polymer Materials in Agriculture and Industry (RPM), Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
b
Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
c
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90363 Lodz, Poland
d
Office of Natural Rubber Research Program, Thailand Science Research and Innovation (TSRI), Bangkok 10900, Thailand
*Correspondence to: Wirasak Smitthipong (E-mail: [email protected])
Abstract Supramolecular structure can be formed using noncovalent interactions based on the self-assembly processes. DNA is a good example for supramolecular materials because it is able to form supramolecular structure by forming specific hydrogen bonds between its base pairs. Moreover, DNA as an anionic medium can bind with oppositely charged materials to form complex structures of various shapes and properties. This work is focused on a foam complex that is formed between negatively charged DNA and positive chitosan. Various characterizations —Fourier transformed infrared spectroscopy (FTIR), Small and Wide-angle Xray scattering (SAXS and WAXS), scanning electron microscope (SEM), texture analyzer and differential scanning calorimetry (DSC) — are used to study the properties of this dried scaffold. The FTIR spectra presented the chemical structure of DNA and chitosan. While the SAXS power law decay has revealed that an increasing of chitosan content smoothens the surface of the structure, on the other hand, the roughness is much higher when the DNA content is increased. The melting point of the foam from the DSC scan has been identified. The mechanical property of foam is suitable for the application of scaffold, and there is no cytotoxicity of foam to the cell. It is expected that this type of biomaterial could be used in several applications such as functional material and as a drug delivery material. Keywords: DNA; chitosan; scaffold; biomaterials; self-assembly; supramolecular materials
Introduction Nucleic acids especially DNA is known for its biological function mainly as storage of genetic information. However, it can be also viewed as a biopolymer with nucleotides building blocks,
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connected together, form a double stranded helix. Due to its recognition potential artificial DNA molecules are being developed into new smart therapeutics. For that natural or modified DNA fragments have to be delivered inside cell, crossing hydrophobic part of the membrane. Such highly polar biopolymer is commonly encapsulated or otherwise complexed to screen negative charges. DNA can form complex structures with positively charged materials such as Lipofectamine, polyethyleneimines, positive dendrimers and natural polymers such as chitosan. Chitosan is one of the most abundant polymers found in nature. This linear polysaccharide consists of D-glucosamine and N-acetyl-D-glucosamine is formed during enzymatic deacetylation of chitin. Due to its many advantages, such as biocompatibility, biodegradability, low toxicity, biological activities, and adsorption properties chitosan is considered as efficient drug delivery carrier [1-4]. It has been applied in different forms (solutions, gels, films or fibers) [5]. DNA has been used before as a structural medium for preparation of biofilms in complex with cationic lipid, didodecyldimethylammonium bromide (DDAB) [6-8]. Results suggest that the properties of such biofilms depend on the length of DNA and the nucleic acid-lipid films have lamellar multilayered structure with layers of nucleic acids being separated by lipid bilayers of DDAB [6]. The supramolecular self-assembling films between random coil poly(styrene sulfonate) mix with DDAB (PSS-DDAB films) and double stranded DNA mix with DDAB (DNADDAB films) have been investigated [9]. It appears that DNA-DDAB film has more mechanical properties than PSS-DDAB film. So, the chain length and molecular structure of nucleic acid can also affect the mechanical and morphological properties of the films. Increase the DNA content also increases the tensile strength of the films and has more lamellar multilayer structure. The complex of DNA with chitosan was studied before, however exclusively in solution by varying the fractional content of acetylated units (FA) and degree of polymerization (DP) of chitosan [10]. The properties of DNA-chitosan polyplexes depend strongly on the FA, charge density and structure of chitosan. Increasing FA was found to increase the size and reduce the compactness of the polyplexes. The stability of polyplexes depended strongly on DP and pH, the complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. This literature work focused on stability properties of polyplexes in the aqueous environment, however, the relationship between the resulting structures and properties of DNA:chitosan material remains to be investigated. The main goal of this work was to prepare and analyze physicochemical and mechanical properties of DNA:chitosan scaffolds at different composition ratios in the dry state, calculated based on the stoichiometry of electrostatic interaction. We have generated the DNA:chitosan scaffold using the in-house developed freezedry procedure, which allow us to control the mechanical and surface properties of these scaffolds. Experimental Materials The mixture of DNA with chitosan was prepared at different molar ratios of 0.25:1, 0.5:1, 1:1, 1:0.5 and 1:0.25 DNA and chitosan respectively. Typically, a DNA sample (deoxyribonucleic acid sodium salt from salmon testes, 2000 base pairs from Sigma-Aldrich) was dissolved in 5 mL of distilled water and stirred for 30 min. The chitosan (MW ~15,000 Da from Polysciences, Inc.) was dissolved in 5 mL of 1% acetic acid and stirred for 30 min. The chitosan solution was mixed with DNA solution and stirred vigorously at room temperature for 3 h. Mixed samples were washed three times in distilled water, placed in a freezer at -20°C for 24 h followed by drying for 24 h in a freeze dryer.
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Measurements DNA:chitosan complex samples ratio of 0.25:1, 0.5:1, 1:1, 1:0.5 and 1:0.25 were tested by elemental analysis for carbon, hydrogen and nitrogen atoms (LECO CHNS-932). Structures were subjected to Small and Wide-angle X-ray scattering analysis (SAXS and WAXS) using a custom-made instrument at the x-ray diffraction facility in the Materials Research Laboratory (MRL) at UCSB, USA. The instrument utilizes a 50 µm microfocus Cu target x-ray source with a parallel beam multilayer optics and monochromator (Genix from XENOCS SA, France), high efficiency scatterless hybrid slits collimator developed in house, and Pilatus100k and Eiger 1M solid state detectors (Dectris, Switzerland). Data reduction and model fitting were carried using the NIKA and IRENA software packages developed at Argonne National Laboratory. Composition of complexes was analyzed by Fourier transform infrared spectroscopy (FTIR) using ATR-FTIR mode (BRUKER, The VERTEX 70 of the Bruker VERTEX Series) with 32 scans and wavelength range 4000-400 cm-1. Differential scanning calorimetry (DSC) (Perkin Elmer) has been employed to get thermal profile of mixed DNA:chitosan samples. All complexes were heated from 25°C to 200°C, the heating scan was performed at a scan rate of 10°C/min. Morphological structure of dried complexes was established with scanning electron microscope (SEM) model Quanta 450 FEI with tungsten filament as an electron source and typical magnification 50x. Then the micrographs from the SEM were analyzed by the ImageJ software to determine the porosity and pore size of the scaffold. Compression test was conducted by the Texture Analyzer Model TA.XTplus, using 2 mm probe with the speed of 0.03 mm/s at the depth distance of 3 mm from surface of the sample. The scaffold sample was prepared into a mold (cylindrical shape with 3 cm in diameter and height). Each of the samples was tested at least three times. Cytotoxicity of complexes was measured using standard XTT assay in aortic valve endothelial cells isolated from porcine heart valves and cultured in the laboratory. Dried complex samples of DNA:chitosan were sterilized by exposure to the UV light for 6 h. Then 1400 µL of cEGM2 (culture medium) was added into each microtube containing dry samples. A sample of conditioned media (CM-C) was used as a control. All samples CM-R1, CM-R1, CM-R2, CM-R3, CM-R4, CM-R5 corresponding to the molar ratio 0.25:1, 0.5:1, 1:1, 1:0.5 and 1:0.25, respectively, and control CM-C were incubated in the 5% CO2 at 37°C for 48 h. After the incubation all media samples were sterilized by filtering through 0.2 µm filters under the sterile condition. Porcine aortic valve endothelial cells were collected and seeded in 96-well plate at 2,500 cells per well and cultured overnight in low serum media (0.4% FBS EGM2). Then cells were incubated in conditioned CM-C and CM-R1 to CM-R5 media samples for 48 h. After 48 h incubation standard XTT assay was carried according to manufacturer’s protocol. Cell toxicity measurements were done in triplicates (n = 3). Statistical analysis was done with Kruskal-Wallis test (using ANOVA for non-parametric data). P value of P<0.05 was considered to be statistically significant. Results and Discussion The DNA:chitosan complex has been developed and studied before for targeted DNA fragments delivery into cells under the aqueous environment. Since chitosan is a natural product with biocompatibility and negligible cytotoxicity, this approach creates an interesting alternative for
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artificial polymers. Here the DNA and chitosan mixture of various ratios was obtained in the form of solid porous material in the dry state (Figure 1). This foam type of material was obtained by in-house developed freeze-dry procedure. The stoichiometry used in this study ranged from 0.25:1 to 1:0.25 for DNA and chitosan respectively, however the sample of 0.25:1 before drying looked different. As all remaining samples had semi-liquid composition otherwise known as “complex coacervation” [11], but the one with the lowest DNA content precipitated during preparation (Figure S1). In simple coacervation process, complex structure can be occurred by the electrostatic interaction between positive charge and negative charge polymers with optimum ionic strength [12, 13]. Due to chitosan is well-known as having a good complex ability [3] and has many functional groups on their back bone [1]. So, chitosan can interact with the negative charge DNA through the electrostatic interaction in order to obtain the self-assemble material. In the present study of DNA:chitosan (0.25:1) mixture, only the small content of DNA can be represented the complex precipitation. Beyond this content of DNA, the complex coacervation between higher DNA content and chitosan is obtained. To determine the theoretical charge ratio of DNA to chitosan we estimated for DNA to be 325g/base and therefore per charge and for chitosan to be 161g/unit. With this estimation the theoretical ratio for 1:1 charge would be in the sample of 0.5:1 for DNA:chitosan mixture. For other samples the remaining charges in the mixture are screened by residual counterions. After that, all the complex samples were washed three times in distilled water, placed in a freezer at -20°C for 24 h followed by drying for 24 h in a freeze dryer. Finally, the DNA:chitosan scaffolds are obtained (Figure 1). All scaffold samples were first tested for its composition using elemental analysis, Fourier transformed infrared spectroscopy and glass transition melting temperature. Elemental analysis of carbon, hydrogen and nitrogen atoms in the DNA:chitosan dried complex for selected samples was compared with theoretical values (Table 1).
Figure 1 Picture of scaffold foam type material obtained from the mixture of DNA with chitosan in the ratio of 0.25:1, 0.5:1, 1:1, 1:0.5 and 1:0.25 respectively.
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Some discrepancy in results can be associated with the DNA composition, which consists of four types of nucleobases with different element contents. Differences might be also due to the remaining counter-ions mainly from the chitosan, as DNA was used in the sodium salt form. Nonetheless the composition of hydrogen and nitrogen seem to be correct. DNA has four types of base pairs and each base pair has different element contents. Table 1 Elemental analysis of DNA:chitosan complexes in ratios 0.5:1, 1:1 and 1:0.5 (C: carbon atom, H: hydrogen atom and N: nitrogen atom). DNA:chitosan ratio
a
Theoretical result (wt%) a
Experimental result (± 1 wt%)
C
H
N
C
H
N
0.5:1
35
5
7
33
6
9
1:1
39
5
11
31
5
14
1:0.5
38
5
12
30
5
13
Theoretical elemental of DNA:chitosan by calculation.
Fourier transformed infrared spectroscopy spectra were collected for pure samples of DNA and chitosan (Figure S2, S3) and results are in good agreement with those present in the literature [5, 13]. When DNA was mixed with chitosan the FTIR spectral analysis shows characteristics for both species (Figure 2). Bands at 3353, 3218 and 2876 cm-1 are associated with the -OH stretching vibration of pyranose ring, the NH stretching vibration in primary amide and to the CH asymmetric stretching vibration in the chitosan ring, respectively. Bands at 1651, 1586 and 1376 cm-1 are characteristic for N-acetyl groups with the C=O, stretching vibration in primary amide, the NH bending in secondary amide and CN stretching vibration in tertiary amide, respectively. The absorption band at 1416 cm-1 corresponds to amino groups. The symmetrical deformation for CH3 can be observed at 1316 cm-1. The vibration C-O-C glycosidic linkage is observed at 1151 cm-1. The sugar phosphate backbone vibration range with the asymmetric stretching of phosphate groups can be found at 1220 cm-1, the symmetric stretching of phosphate groups at 1060 cm-1 and C-C stretching vibration (deoxyribose) at 964 cm-1. The signal at 1018 cm-1 suggests that DNA adopts B type conformation [7]. It can be noticed that multiple characteristic peaks in FTIR spectra for DNA are partially separated from peaks for chitosan, thus it is easy to ascertain that the mixture composes both molecules, however, it is impossible to correlate the signal with the composition based on this analysis. Differential scanning calorimetry (DSC) was used to monitor heat effects associated with phase transitions and physical properties of scaffold as a function of temperature and also establish melting temperatures for all DNA:chitosan mixture complexes (Figure S4). Distinctive endothermic peak in the melting temperature (Tm) for dried complexes can be found in 5 mixtures (from 0.25:1 to 1:0.25). DSC values collected in Table 2 show gradual increase of the melting temperature with the increase of the DNA content. This might be due to the approximate size of DNA molecules as compared to chitosan (DNA molecules for 2,000 base pairs is approximately 1,300 kDa and chitosan is 15 kDa). Also, the double helix DNA structure might contribute to the thermal stability of the complex. This result is similar to the previous studies, a board endothermic peak, for solid DNA and DNA in solution [14-17], is observed which corresponds to DNA melting.
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1219-962 cm -1 1710-1484 cm -1
A 1416-1376 cm -1
B
Absorbance
C
D
E
F
G 3500 3300 3100 2900 2700
Wavenumber (cm-1)
1800
1600
1400
1200
1000
800
Wavenumber (cm-1)
Figure 2 The FTIR spectra of (A) DNA alone, (B) chitosan alone, (C) DNA:chitosan = 0.25:1, (D) DNA:chitosan = 0.5:1, (E) DNA:chitosan = 1:1, (F) DNA:chitosan=1:0.5 and (G) DNA:chitosan = 1:0.25. Table 2 Melting point and glass transition temperature in the DNA:chitosan ratios from 0.25:1, to 1:0.25. DNA:chitosan ratio
Melting temperature (± 1°C)
0.25:1 0.5:1 1:1 1:0.5 1:0.25
60 70 73 72 72
Once the composition of the DNA:chitosan complex was established and melting temperature profile suggests that homogeneous mixture is formed, we were curious how the structure looks like and whether it is influenced by ratio change. For structural analysis the Small and Wide-angle X-ray scattering (SAXS and WAXS) and scanning electron microscopy (SEM) were used. The length scale range for SAXS and WAXS analysis was 0.1 nm - 100 nm (Figure 3). The SAXS region corresponds to the larger scale structures (up to 100 nm), and the
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main characteristics here can be described by Porod scattering. In the general power-law decay, linear Log(I) vs Log(Q) the intensity can be explained as 1/QP, where P is the Porod parameter or power law exponent. This parameter correlates to the geometry and interface roughness of the scattering objects and thus can be used to differentiate between different structures. The results show some differences in the SAXS analysis of different DNA:chitosan ratios. The dried complexes in ratios of 0.25:1, 0.5:1 and 1:1 show similar scattering behaviour, with a power law decay ~1/Q4 suggesting smooth surface of samples. However, dried complexes in the ratio 1:0.5 and 1:0.25 show scattering behaviour, with the power law decay ~1/Q3, suggesting the rough surface structure. Essentially when the chitosan content is high the surface is rather smooth and when DNA content is increased the roughness much higher. High Porod parameters are usually assigned to the large 3-dimensional structures (>100 nm). In other words, what we are seeing is not the size of the object, but rather the surfaces within. Additionally, there are no obvious diffraction peaks present in the data, which indicates that there is no ordering at the large length scale which SAXS probes [18-20]. Concurrently the WAXS experiment shown different profile depending on the DNA:chitosan ratio. Individually both, DNA and chitosan have been investigated by X-ray diffraction previously [21, 22]. In the DNA:chitosan complexes, with ratio close to 1:1 we can recognize two groups of signals; at Q ≈ 1.85 A-1 for DNA and at Q ≈ 0.75 A-1 for chitosan. The DNA:chitosan complexes at ratios 0.25:1 and 1:0.25 present broad peaks with no distinctive characteristics [18-20]. However, beyond these basic descriptions of the data, we do not have a good solid model to explain the features because the scaffold structure could be quite complex. In anyway, Tirrell and co-workers have successfully prepared the supramolecular self-standing film based to electrostatic interaction between anionic DNA and cationic amphiphile (DDAB) [4]. They found that the SAXS data for DNA-DDAB sample shows similar harmonic peaks, suggesting a lamellar-like structure with layers of DNA being separated by lipid bilayers of DDAB. This result is in agreement with the results obtained previously by Safinya [10]. However Tirrell’s samples were dried samples, whereas Safinya’s samples were analyses in the aqueous solution. In the previous work, we investigated the supramolecular film between 100 nucleotides long poly(adenylic acid) and 15-30 nucleotides short RNA [4]. We found that the film’s morphology changes (analyzed by AFM), height images show a rougher surface when we increase the concentration of long poly(adenylic acid) in the mixture and plateau at around 25 wt% of poly(A).
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Figure 3 SAXS and WAXS analysis of DNA:chitosan dried samples in five ratios, from 0.25:1 to 1:0.25. SAXS data (upper Figure) represents short scale of Q [A-1] whereas WAXS data (lower Figure) represents longer scale of Q [A-1]. In the SAXS region, the Porod model of general power-law decay (1/QP where P is called the Porod parameter) can be applied. The Porod parameter of 4 and 3 can be interpreted as the dried complexes structure had a smooth and rough surface structure, respectively. The Porod parameter of 3 was better order then the Porod parameter of 4. In the WAXS region (lower Figure), there are two groups of peak which are from DNA (Q ≈ 1.85 A-1) and chitosan (Q ≈ 0.75 A-1) separately. Morphological characteristics were acquired by SEM (Figure 4). From the first glance we can see that all dried complexes have a porous-type structure. This is very promising in the view that these dried complexes might be used as a scaffold. Pore size is a very important factor for mechanical, elastic, cell adhesion, migration and proliferation properties. It can be divided into nano-size (nano-roughness, <100 nm), micro-size (micro-roughness, 100 nm - 100 µm) and macro-size (100 µm – 1 mm). The porosity for obtained structures was quantified based on the ImageJ contrast analysis (Figure 4 right).
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Figure 4 SEM analysis (magnification 50x) of DNA:chitosan samples in five ratios, from 0.25:1 to 1:0.25 from A to E respectively: (left panel) presents examples of SEM images as collected during analysis, (right panel) presents the two-color scale contrast as it was used for porosity analysis by ImageJ software.
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Table 3 Pore size and percent porosity for five dried DNA:chitosan complex samples analyzed by ImageJ software. Porosity (± 1 %) Pore size (± 1 µm) DNA:chitosan ratio 0.25:1 0.5:1 1:1 1:0.5 1:0.25
105 – 1294 121 – 650 75 – 447 98 – 845 144 – 937
62 60 58 51 49
The results of pore size and percent of porosity for all 5 dried samples shown large differences in the pore size, ranging from macron sizes, 100 µm up to 1 mm. In the scaffold to be considered as biomaterial for cell growth or bio-mineralization, the macro-pore size plays an important role. For instance, cell seeding, cell distribution, migration, differentiating and further neo-vascularization in vivo is dependent on the material’s surface properties and also on the porosity [23]. The porosity is measured in the black areas compared to the total areas (Figure 4) using the ImageJ software, it was found that porosity of scaffold tends to decrease with increasing of DNA content. This could be explained by the fact that DNA molecule is bigger than chitosan molecule (DNA molecule is 2000 base pairs or ≈ 1300 kDa while chitosan molecule is 15 kDa). Moreover, the structure of DNA molecule is a double helical structure while the structure of chitosan molecule is a linear polysaccharide. So, DNA molecule can affect the porosity of the dried complexes. The highest homogeneity in pore size has a 1:1 complex, however those from a ratio of 0.5:1 to 1:0.5 can be considered for cell growing, generating and differentiating the cells into organs or cardiovascular supportive structure. Although, we observed small differences in the SEM images, these probably would not count for the differences between low and high DNA content in samples observed in SAXS. These are most probably manifestation of fine nanostructure organization. We also studied the mechanical properties of the scaffolds for further use in materials applications such as tissue engineering, etc. Compressive strength values for each type of scaffold were determined at least in triplicate. Statistics on a completely randomized design were determined using Statgraphic software (release 5.0, Statgraphic Corp.) to calculate analysis of variance (ANOVA). Bonferroni’s multiple range test was used to determine significantly different averages at a 95% confidence interval [24]. We found that the compressive strength of scaffolds increases with increasing of DNA content (Table 4). This result is in good agreement with the melting point and morphological properties of the scaffolds, DNA molecule could also affect the mechanical properties of this type of scaffold. So, we can design and develop a tailor-made scaffold for the specific application, in particular the DNA:chitosan scaffold with optimum mechanical properties. Regarding to a soft material as foam rubber, its compressive strength can be varied during 0.01-30 MPa depending on the foam formulation and density [25, 26]. Generally, scaffold should provide a mechanical strength in the range of 0.05-350 MPa. For example, the moduli of poly(ethylene glycol)-terephthalate scaffolds were 0.05-2.5 MPa in dry state [27]. Table 4 Compressive strength for five dried DNA:chitosan scaffold ratios from 0.25:1 to 1:0.25. DNA:chitosan ratio
Compressive strength (± 0.01 MPa)
0.25:1 0.5:1
0.20 0.35
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DNA:chitosan ratio
Compressive strength (± 0.01 MPa)
1:1 1:0.5 1:0.25
0.56 0.54 0.52
In anticipation of this material to be used as a biocompatible platform to grow cell and tissue we want to investigate the cytotoxicity and cell compatibility. The most important is whether prepared complexes will release toxic material into medium. For that DNA:chitosan samples were treated with cell growth media and then XTT cytotoxicity test was performed after 48 h incubation. We used porcine aortic valve endothelial cells (PAVEC) as a model cellular system. Results shows no cytotoxicity, actually one can notice slight increase in proliferation after incubation with DNA:chitosan complex (Figure 5). The complex composition has also very small effect on cell viability, with the statistical threshold setup at P<0.05 there is no statistical difference between CM-R1 to CM-R5 samples.
Figure 5 Cell cytotoxicity measurement using XTT test after 48 h porcine aortic valve endothelial cells treatment with DNA:chitosan complex samples CM-R1 to CM-R5 of 0.25:1 to 1:0.25 DNA to chitosan respectively. Conclusions We demonstrate efficient method of coacervation complex preparation using anionic DNA and cationic chitosan in ratios ranging from 0.25:1 to 1:0.25 (DNA to chitosan). During desiccation all samples form foam scaffold type of structure. The composition of dried samples was confirmed using elemental analysis, Fourier transformed infrared spectroscopy and differential scanning calorimetry. The structure of newly created scaffold was analyzed with Small and Wide-angle X-ray scattering (SAXS and WAXS) and scanning electron microscopy (SEM). Surprising to us was that when the chitosan content is high (ratio 0.25:1, 0.5:1 and 1:1) the surface is rather smooth and when DNA content is increased (ratio 1:0.5 and 1:0.25) the roughness is much higher, as suggested by SAXS power law decay. As SEM analysis indicates, structures of all dried complexes have relatively large-scale pores (100 - 1000 µm), however, percentage porosity decrease with increase of DNA content in the sample. The mechanical property of foam is suitable for the application of scaffold. Very interesting for us was to find out that all samples have no effect on cell toxicity and proliferation. This was tested using primary porcine aortic valve endothelial cells, it would be interesting to see similar results on other cell lines especially those where contact with implants and artificial scaffold is anticipated. In
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perspective, these types of material could be useful for medical application as a functional material, and drug delivery system. Acknowledgements The authors gratefully acknowledge the financial support of the Kasetsart University Research and Development Institute (KURDI), Bangkok, Thailand. Moreover, the authors particularly grateful for the assistance given by Dr. Youli Li and Mr. Phillip Kohl for SAXS and WAXS measurements from Materials Research Laboratory (MRL), University of California, Santa Barbara, USA. The SAXS and WAXS research carried out here made extensive use of shared experimental facilities of the MRL: an NSF MRSEC, supported by NSF DMR 1720256. The MRL is a member of the NSF-supported Materials Research Facilities Network (www.mrfn.org). Data availability The raw/processed data required to reproduce these findings cannot be shared at this time due to legal or ethical reasons.
References and notes [1] S. Kim, M.E. Nimni, Z. Yang, B. Han, Chitosan/gelatin–based films crosslinked by proanthocyanidin, Journal of Biomedical Materials Research Part B: Applied Biomaterials 75B(2) (2005) 442-450. [2] R. Jayakumar, N. Nwe, S. Tokura, H. Tamura, Sulfated chitin and chitosan as novel biomaterials, International Journal of Biological Macromolecules 40(3) (2007) 175-181. [3] M.M.F.A. Baig, M. Naveed, M. Abbas, W. Chunxia, S. Ullah, M. Hasnat, A. Shad, M. Sohail, G.J. Khan, M.T. Ansari, DNA scaffold nanoparticles coated with HPMC/EC for oral delivery, Int J Pharm 562 (2019) 321-332. [4] P. Pakornpadungsit, W. Smitthipong, A. Chworos, Self-assembly nucleic acid-based biopolymers: learn from the nature, Journal of Polymer Research 25(2) (2018) 45. [5] M. Rinaudo, Chitin and chitosan: Properties and applications, Progress in Polymer Science 31(7) (2006) 603-632. [6] W. Smitthipong, T. Neumann, A. Chworos, L. Jaeger, M. Tirrell, Supramolecular Materials Comprising Nucleic Acid Biopolymers, Macromol. Symp. 264 (2008) 13-17. [7] R. Chollakup, W. Smitthipong, Chemical structure, thermal and mechanical properties of poly(nucleotide)–cationic amphiphile films, Polymer Chemistry 3(9) (2012) 2350-2354. [8] S. Yasmeen, M. Kabiraz, B. Saha, M. Qadir, M. Gafur, S. Masum, Chromium (VI) Ions Removal from Tannery Effluent using Chitosan-Microcrystalline Cellulose Composite as Adsorbent, 2016. [9] R. Chollakup, W. Smitthipong, A. Chworos, Supramolecular cooperative-assembly of polyelectrolyte films, RSC Advances 3(14) (2013) 4745-4749. [10] S.P. Strand, S. Danielsen, B.E. Christensen, K.M. Vårum, Influence of Chitosan Structure on the Formation and Stability of DNA−Chitosan Polyelectrolyte Complexes, Biomacromolecules 6(6) (2005) 3357-3366. [11] R. Chollakup, W. Smitthipong, C.D. Eisenbach, M. Tirrell, Phase Behavior and Coacervation of Aqueous Poly(acrylic acid)−Poly(allylamine) Solutions, Macromolecules 43(5) (2010) 2518-2528.
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[12] H. Bohidar, Coacervates : A novel state of soft matter - An overview, J. Surface Sci. Technol 24 (2008) 105-124. [13] A.S. Hoffman, Hydrogels for biomedical applications, Advanced Drug Delivery Reviews 64 (2012) 18-23. [14] J.G. Duguid, V.A. Bloomfield, J.M. Benevides, G.J. Thomas, Jr., DNA melting investigated by differential scanning calorimetry and Raman spectroscopy, Biophys J 71(6) (1996) 33503360. [15] Y.S. Tarahovsky, V.A. Rakhmanova, R.M. Epand, R.C. MacDonald, High temperature stabilization of DNA in complexes with cationic lipids, Biophys J 82(1 Pt 1) (2002) 264-273. [16] S.L. Lee, P.G. Debenedetti, J.R. Errington, B.A. Pethica, D.J. Moore, A Calorimetric and Spectroscopic Study of DNA at Low Hydration, The Journal of Physical Chemistry B 108(9) (2004) 3098-3106. [17] Y. Liu, F. Tan, Calorimetric studies of thermal denaturation of DNA and tRNAs, Journal of thermal analysis 45(1) (1995) 35-38. [18] Y. Li, R. Beck, T. Huang, M.C. Choi, M. Divinagracia, Scatterless hybrid metal-single-crystal slit for small-angle X-ray scattering and high-resolution X-ray diffraction, Journal of Applied Crystallography 41(6) (2008) 1134-1139. [19] J. Ilavsky, P.R. Jemian, Irena: tool suite for modeling and analysis of small-angle scattering, Journal of Applied Crystallography 42(2) (2009) 347-353. [20] J. Ilavsky, Nika: software for two-dimensional data reduction, Journal of Applied Crystallography 45(2) (2012) 324-328. [21] C.R. Safinya, Structures of lipid–DNA complexes: supramolecular assembly and gene delivery, Current Opinion in Structural Biology 11(4) (2001) 440-448. [22] G.L. Clark, A.F. Smith, X-ray Diffraction Studies of Chitin, Chitosan, and Derivatives, The Journal of Physical Chemistry 40(7) (1935) 863-879. [23] I. Bružauskaitė, D. Bironaitė, E. Bagdonas, E. Bernotienė, Scaffolds and cells for tissue regeneration: different scaffold pore sizes—different cell effects, Cytotechnology 68(3) (2016) 355-369. [24] R. Chollakup, J. Suesat, S. Ujjin, Effect of Blending Factors on Eri Silk and Cotton Blended Yarn and Fabric Characteristics, Macromolecular Symposia 264 (2008) 44-49. [25] R. Suksup, Y. Sun, U. Sukatta, W. Smitthipong, Foam rubber from centrifuged and creamed latex, Journal of Polymer Engineering 39 (2019). [26] Y. Zhu, G. Luo, R. Zhang, Q. Liu, Y. Sun, J. Zhang, Q. Shen, L. Zhang, Investigation of the Constitutive Model of W/PMMA Composite Microcellular Foams, Polymers 11(7) (2019). [27] V. Vishwanath, K. Pramanik, A. Biswas, Optimization and evaluation of silk fibroin-chitosan freeze-dried porous scaffolds for cartilage tissue engineering application, Journal of Biomaterials Science, Polymer Edition 27(7) (2016) 657-674.
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DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold Pitchaya Pakornpadungsita, Thridsawan Prasopdeea, Napachanok Mongkoldhumrongkul Swainsonb, Arkadiusz Chworosc, Wirasak Smitthiponga,d a
Specialized Center of Rubber and Polymer Materials in Agriculture and Industry (RPM), Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand c Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90363 Lodz, Poland d Office of Natural Rubber Research Program, Thailand Science Research and Innovation (TSRI), Bangkok 10900, Thailand Correspondence to: Wirasak Smitthipong (E-mail: [email protected]) Highlights: • • • • •
Porous scaffold can be obtained by self-assemble complex between DNA and chitosan. Composition of dried scaffolds was confirmed by elemental analysis, FTIR and DSC. Structure of newly created scaffold was analyzed by SAXS & WAXS and SEM. Mechanical property of foam is suitable for the application of scaffold. Scaffold samples have no effect on cell toxicity and proliferation.
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DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold Pitchaya Pakornpadungsita, Thridsawan Prasopdeea, Napachanok Mongkoldhumrongkul Swainsonb, Arkadiusz Chworosc, Wirasak Smitthiponga,d a
Specialized Center of Rubber and Polymer Materials in Agriculture and Industry (RPM), Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand c Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90363 Lodz, Poland d Office of Natural Rubber Research Program, Thailand Science Research and Innovation (TSRI), Bangkok 10900, Thailand Correspondence to: Wirasak Smitthipong (E-mail: [email protected]) Conflicts of Interest: The authors declare no conflict of interest in reported research. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
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DNA:chitosan complex, known as a drug delivery system, can create a porous scaffold Pitchaya Pakornpadungsita, Thridsawan Prasopdeea, Napachanok Mongkoldhumrongkul Swainsonb, Arkadiusz Chworosc, Wirasak Smitthiponga,d a
Specialized Center of Rubber and Polymer Materials in Agriculture and Industry (RPM), Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand b Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand c Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90363 Lodz, Poland d Office of Natural Rubber Research Program, Thailand Science Research and Innovation (TSRI), Bangkok 10900, Thailand Correspondence to: Wirasak Smitthipong (E-mail: [email protected]) Author statement: P.P. carried out the preparation and characterization of scaffold; T.P. carried out the SAXS and WAXS; N.M.S. carried out the cytotoxicity assay; A.C. contributed to the discussion and review of the manuscript; W.S. conceived the study, designed the study and helped with the manuscript. This manuscript and its contents, has not been published previously and is not under any consideration for publication in any other journal at the time of this submission. All authors gave final approval for publication and agree to be held accountable for the work performed therein.
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