Bacterial Cellulose Nanocrystals (BCNC) Preparation and


Dec 6, 2017 - Bacterial cellulose (BC) is an excellent renewable resource of high-purity cellulose that can be used as original fiber forms or isolate...
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Bacterial Cellulose Nanocrystals (BCNC) Preparation and Characterizations from Three Bacterial Cellulose Sources, and Development of Functionalized BCNC as Nucleic Acid Delivery Systems Pratyawadee Singhsa, Ravin Narain, and Hathaikarn Manuspiya ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00105 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Bacterial Cellulose Nanocrystals (BCNC) Preparation and Characterizations from Three Bacterial Cellulose Sources, and Development of Functionalized BCNC as Nucleic Acid Delivery Systems Pratyawadee Singhsa†,‡, Ravin Narain‡,*, Hathaikarn Manuspiya†,* †

The Petroleum and Petrochemical College, Center of Excellence on Petrochemical and

Materials Technology, Chulalongkorn University, Soi Chulalongkorn 12, Pathumwan, Bangkok 10330, Thailand. ‡

Department of Chemical and Materials Engineering, Donadeo Innovation Centre for

Engineering, 116 Street and 85 Avenue, Edmonton, AB T6G 2G6, Canada. KEYWORDS: Bacterial cellulose, Bacterial cellulose nanocrystals, Acid hydrolysis, Crystallinity, Cationic surface modification, Cytotoxicity, Nucleic acid delivery

ABSTRACT: Bacterial cellulose (BC) is an excellent renewable resource of high purity cellulose which can be used as original fiber forms or isolated nanocrystalline forms, known as bacterial cellulose nanocrystals (BCNC) which have gained more attention in the development of highly biocompatible biomaterials. In this work, the BCNC production was studied about the influences

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of the varying BC sources (BC pellicles produced by three different BC-producing bacterial strains of Komagataeibacter xylinus) and acid hydrolysis conditions (hydrochloric acid, sulfuric acid, and mixture of both acids) on the production yield, morphology, and physicochemical properties of the resulting BCNC. The BCNC production with these variable factors provided the distinctive characteristics rod-like nanocrystals which can be useful for various applications. For demonstration of the biomedical application as nucleic acid delivery systems, the cationic BCNC were developed by the simple cationic surface modification by the physical adsorption of the obtained sulfuric hydrolyzed BCNC with amines and amine-containing polymers, and the resulting cationic modified BCNC were evaluated their complexation ability with siRNA and cytotoxicity in HeLa cells. All unmodified and cationic modified BCNC samples exhibited low toxicity at the concentrations of 0.1 mg/mL which assured their good biocompatibility, and the cationic modified BCNC with methacrylamide polymers were fully complexed with siRNA. Therefore, this research suggested that the BCNC with desired properties can be produced by selecting the proper BC sources and acid conditions, also the cationic functionalized BCNC which revealed the potential as nucleic acid nanocarriers were easily prepared by the simple cationic surface modification.

Introduction Cellulose is a highly available natural biopolymer which has become a good candidate in development of biomaterials with required characteristic features of renewability, sustainability, and biocompatibility. Cellulose can be obtained from several sources such as plants, some algae species and microbes (bacteria).1 Particularly, cellulose produced from bacteria which is known as bacterial cellulose (BC) has been more attractive due to its higher purity not containing other

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kinds of biopolymers, i.e. hemicellulose, pectin, and lignin which generally found in plantderived cellulose.2 BC is synthesized by various bacterial species, the most effective cellulose producing specie which has often been used as a model organism in BC production studies is Komagataeibacter xylinus (formerly Gluconacetobacter xylinus and Acetobacter xylinum).3 BC has distinct ribbon-like 3D network structure (around 100 nm in diameter and around 100 µm in length) which is composed of extremely fine cellulose fiber, microfibrils of 2-4 nm in diameter.4 Moreover, BC presents fascinating properties such as high water holding capacity, high porosity, high mechanical strength, high crystallinity, low toxicity, and biocompatibility, resulting in BC can be utilized in many applications, especially in the fields of biomedicine and pharmacology.5-8 Additionally, BC is a promising starting material for producing cellulose nanocrystals (CNC), or cellulose nanowhiskers, due to its unique characteristics of high purity and high crystallinity.9-13 Bacterial cellulose nanocrystals (BCNC) are rod-like nanocrystals obtained from the isolation or extraction of crystalline regions of bacterial cellulose. The geometrical dimensions of produced BCNC are about 10-50 nm in diameter and 100-1500 nm in length dependent on the sources of BC and the isolation methods.14-15 The main process for the isolation of BCNC form BC fibers is based on acid hydrolysis. Amorphous or disordered sections of cellulose are better hydrolyzed by hydrogen ions, enhancing the hydrolytic cleavage of the glycosidic bonds that releases individual crystallites of remaining intact crystalline regions which have a higher acid resistance.16 Several inorganic acids, i.e. hydrochloric (HCl), sulfuric (H2SO4), phosphoric, and hydrobromic acids have been used in acid hydrolysis of cellulose; however, HCl and H2SO4 are more regularly used ones.10 The HCl hydrolysis provides the nanocrystals with the low-density charges on their surface, resulting in the limitation of nanocrystal dispersibility and increase in flocculation of their aqueous suspensions.17 Whereas, the H2SO4 hydrolysis generates the highly

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negatively charged nanocrystals by the sulfonation of the surface hydroxyl groups of cellulose, consequently, the highly stable colloidal suspensions can be obtained.18-19 However, the introduction of surface sulfate groups can cause the decrease in the thermal stability of the resultant nanocrystals.20-21 Interestingly, the combination of both HCl and H2SO4 acids could tune the surface charge density and improve the thermal stability of the nanocrystals.22-23 According to appealing intrinsic characteristics of cellulose nanocrystals such as nanoscale dimensions, unique morphology, low density, large specific surface area, high aspect ratio, high degree of crystallinity, and high mechanical strength, they have been greatly used as reinforcing fillers in nanocomposite materials.24-26 Furthermore, CNC are highly hydrophilic, biocompatible, and biodegradable which facilitate their usefulness in a variety of biomedical applications including tissue engineering,27-28 bioimaging/biosensing,29-30 and drug delivery.31-34 In addition, it is a significant advantage of using rod-like CNC as drug carriers because such nanoparticles were found to be cell internalized faster than spherical nanoparticles.35-36 Due to their outstanding properties, the rod-like CNC were also expected to replace carbon nanotubes which are well-known that they have excellent penetrability and encapsulation efficiency for drug and gene delivery but poor biocompatibility.37-39 Particularly, the surface of CNC has an abundance of hydroxyl groups which can be easily modified to other functional groups for non-covalent and covalent attachment of biomolecules. As consequence, the facile nanocarriers for targeted drug delivery applications can be developed by the surface modification of cellulose nanocrystals. For a purpose of potential biomedical applications, cationic-modified CNC have been recently attractive owing to their ability to interact with anionic active substances for drug delivery,37, 40 also negatively charged nucleic acids for gene delivery.37, 41 The cationic surface modification of CNC can be accomplished by attaching small molecules or polymers via non-covalent or

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covalent interactions. There are few reports about cationization of CNC, which most of them have been performed by the chemical modification,41-44 including the nucleophilic addition of the activated hydroxyl groups on cellulose to the epoxy moiety of epoxypropyltrimethylammonium chloride (EPTMAC),42 and the cationic polymer grafting by surface-initiated single-electrontransfer living radical polymerization (SI-SET-LRP) method.41 Although highly effective, the chemical modification techniques usually involve tedious and delicate reaction/polymerization procedures, and are inconvenient. In the physical modification, CNC are modified by the physical adsorption via electrostatic interaction, hydrophilic affinity, van der Waals interactions or hydrogen bonding.45-46 In comparison with the chemical modification, the physical approach is a simple and convenient operation and procedure. Therefore, the physical adsorption is an interesting technique for producing cationic CNC. In gene therapeutic applications, RNA interference (RNAi) has emerged as an attractive route where affected gene can be silenced by delivering small interfering RNA (siRNA) to the disease cells.47 However, the delivery of siRNA is challenging due to the problems of enzymatic degradation and immunological responses of naked siRNA, and its negative charge property which limits its penetration ability through negatively charged cell membrane.48 Therefore, nanocarriers are necessary to efficiently deliver siRNA to desired target cells, one of most studied nanocarriers is a non-viral carrier fabricating from synthetic cationic polymers49-52 which have been extensively concerned about their high toxicity.53-54 According to that, new delivery nanocarriers developed from biomaterials have become very interesting. Owing to the characteristics of nano-sized and rod-like particles with good biocompatibility, the cellulose nanocrystals can be a good candidate for developing an efficient nanocarrier with low cytotoxicity effect for siRNA delivery (nucleic acid delivery).

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Thus, it was fascinating to produce the BCNC with various properties which can be further appropriately selected for any desired applications, by using the varying BC sources (three different BC pellicles) and acid types (hydrochloric acid, sulfuric acid, and mixture of two acids). The structural characteristics of the resulting BCNC were evaluated and reported in terms of morphology, particle size, surface charge, crystallinity, crystallite size, and thermal stability. In addition, to demonstrate the potential of BCNC in biomedical applications, the functionalized (cationic) BCNC as nucleic acid delivery systems were developed by using simple cationic surface modification by physical adsorption with chemicals and polymers containing amine functional groups. The obtained cationic modified BCNC samples were evaluated for their physiochemical characteristics, and their potential as nucleic acid delivery devices by testing the cationic modified BCNC-siRNA complexation as well as the cytotoxicity in HeLa cells. Experimental section Materials. Two strains of Komagataeibacter xylinus (K. xylinus TISTR 975, and 1011) were obtained from Thailand Institute of Scientific and Technological Research, and one K. xylinus was obtained from the Institute of Food Research and Product Development, Kasetsart University, Thailand. D-glucose, yeast extract, peptone, calcium carbonate (CaCO3) were obtained from. Hydrochloric acid (ACS reagent, 37%), sulfuric acid, 2-propanol, and dimethylsulfoxide were purchased from Caledon Laboratory Chemicals. Sodium hydroxide, ethylenediamine (EDA), N,N-dimethyethylenediamine (DM), 3-morpholinopropylamine (MP), and 1-(2-aminoethyl)-piperazine (AEP) were purchased from Sigma-Aldrich. Two of methacrylamide

polymers,

namely

poly(N-[3-(dimethylamino)propyl]methacrylamide

hydrochloride) (p(DMAPMA.HCl65) Mn = 13500 Mw/Mn 1.21) and poly(N-[3-(dimethylamino)propyl]methacrylamide

hydrochloride-co-2-Aminoethyl

methacrylamide

hydrochloride)

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(p(DMAPMA65-b-AEMA76) Mn = 27500 Mw/Mn 1.34) were prepared and characterized as shown in previous work.55 Milli-Q water was used for all the experiments. Bacterial cellulose (BC) production. Each of K. xylinus strains was cultured on glucose yeast extract (GYE) agar containing 100 g D-glucose, 10 g yeast extract, 5 g peptone, 20 g CaCO3, 25 g agar per liter at 30 °C for 3 days. Working cultures were routinely prepared on GYE and stored at 4 °C until use. For media use, the glucose yeast extract broth (GYB) was selected from the literature and modified for the present study. The GYB consists of 50 g glucose and 5 g yeast extract in one-liter solution. Before use, all the media were autoclaved at 121 °C for 15 min. The pH was adjusted to 5.0 with HCl or NaOH. For preparation of each K. xylinus strain seed culture, a single colony from a working plate of GYE agar was selected and inoculated in 10 mL of each of six modified GYB media. These seed cultures were incubated for 7 days at 30 °C under static condition. Following growth, bacterial cells were separated from cellulose pellicles in the seed cultures by vigorously shaking; as a result, the cell suspension for inoculation was obtained. Cultures were grown in 250-mL Erlenmeyer flasks containing 100 mL of media and inoculated with 5% (v/v) of the cell suspension. The BC production was incubated under static condition for 7 days at 30 °C. After incubation, the BC pellicles were harvested from the cultures and rinsed with distilled water to remove any residual media. The BC products were washed with 2% w/v NaOH at 80 °C for 1 hour, and then washed repeatedly with distilled water until a neutral pH was obtained. Bacterial cellulose nanocrystals (BCNC) production. Pre-treatment: The purified BC pellicles were mechanically disintegrated to a cellulose paste using a laboratory blender at 5000 rpm for about 20 min under ambient temperature to pass through a 60-mesh screen. The cellulose paste was compressed to remove most of the absorbed water. Acid hydrolysis: The dried

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cellulose paste was treated with each acid solution in the ratio of 1:10 g/mL with continuous stirring in different conditions as follow; the 4 N HCl solution at reflux temperature for 4 h for the hydrochloric acid hydrolysis (H), the 65% w/w H2SO4 solution at 60 °C for 2 h for the sulfuric acid hydrolysis (S), and the 1:1-ratio mixture of 4 N HCl solution and 65% w/w H2SO4 solution at 60 °C for 2 h for the two-acid combination hydrolysis (HS). The hydrolysis reactions were terminated by adding with an excess (10 fold) of cold deionized water. The acidic solution was removed by successive centrifugation at 12000 rpm for 10 min until the supernatant became turbid. The sediment was collected and dialyzed (MWCO: 12000–14000) against deionized water until reaching neutral pH. After dialysis, the BCNC content was collected by centrifugation for 20 min at 12000 rpm and 10 °C. Post-treatment: The BCNC samples were redispersed in deionized water to obtain the 1% w/w BCNC suspension and ultrasonicated for 5 min in an ice bath with a probe sonicator (MXBAOHENG, FS-450N Ultrasonic homogenizer sonicator processor cell disruptor mixer) at 40% power. The BCNC suspension was freeze dried (Labconco 2.5 L) at −52 °C and 0.05 mbar for 48 h. Dry weight and yield of BC and BCNC. The investigation of BC production was as the dry weight of cellulose within the volume of medium in liter (g/L), while BCNC production was as the dry weight of freeze-dried BCNC compared to the dry weight of starting BC pellicles (% w/w). The dry weight of BC was determined by weighing the dried BC pellicles which were air dried in a desiccator at room temperature for 3 days until reaching constant weight. Fourier-transform infrared spectroscopy (FT-IR). Each BC and BCNC sample was airdried on a glass slide in the form of a thin film. FT-IR spectra were obtained using an ATR Nicolet iD7 FT-IR spectrometer. All spectra were scanned between 4000 and 400 cm−1 with 128 convolutions at a resolution of 4 cm−1. Baselines for each sample spectrum were normalized

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using the Spectrum software. The fα fraction of the samples was calculated by the following equation (1):  = 2.55∝ − 0.32 Where ∝ of cellulose can be calculated as Aα/(Aα +Aβ) and Aα and Aβ are absorbencies at 750 and 710 cm-1 , respectively. X-ray diffraction (XRD). X-ray diffraction diagrams of dried BCNC samples were recorded using a Rigaku Model SmartLab 4800 diffractometer with the CuKα radiation wave length (λ= 1.54 Å), generated at a voltage of 40 kV and a filament emission of 30 mA. Samples were scanned from 5-40° 2θ-range at scan speed of 2°/min and scan step of 0.02°. The crystallinity index (CrI) and crystallite size (CrS) were calculated based on X-ray diffraction measurements. Crystallinity index was calculated from the following equation: CrI% =

 −   × 100 

Where I200 is the overall intensity of the peak at 2θ about 22.7° and Iam is the intensity of the baseline at 2θ about 18°.56 The CrS was determined using Scherrer equation57 as following: CrS =

Kλ β cosθ

Where K is the shape factor (0.9), λ is the X-ray wavelength (1.54 Å), β is the full width at half maximum height (FWHM), and θ is the Bragg’s angle. Electron microscopy. The dried BC samples were sputter coated with platinum in preparation for FE-SEM imaging. The field-emission SEM Hitachi S-4800 model was used operating at accelerated voltage of 5 kV and magnification of 20k. Transmission electron micrographs of BCNC samples were taken in a JEOL 100CX-2 transmission electron microscope at an

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accelerating voltage of 100 kV. Sample solutions (0.02%, w/v) were deposited on a carboncoated grid and allowed to dry followed by staining with 2 wt% aqueous uranyl acetate solution. Particle size and zeta potential measurements. The hydrodynamic particle sizes and zeta potentials of the unmodified and cationically modified BCNC in aqueous solutions (salt free) were measured at 25 °C using a ZetaPlus-Zeta Potential Analyzer (Brookhaven Instruments Corporation). The reported value is an average of 10 measurements. All measurement was conducted in triplicate. Thermogravimetric analysis (TGA). Thermogravimetric (TGA) curves were recorded with a TA Instruments model SDT Q600 TGA/DSC system. The samples (ca. 10 mg) were heated from 30 °C to 600 °C with a heating rate of 10 °C /min under nitrogen atmosphere. Derivative TG curves (DTG) express the weight loss rate as a function of temperature. Cationic surface modification by physical adsorption. The 1 wt% BCNC suspensions of sulfated BCNC (K9-S) were surface cationically modified by the physical adsorption with amines and amine-containing polymers as illustrated in Scheme 1. Four structural different amines: ethylenediamine (ED), N,N-dimethylethylenediamine (DM), 3-morpholinopropylamine (MP), and 1-(2-aminoethyl)-piperazine (AP) and two methacrylamide polymers containing amine functional groups: p(DMAPMA.HCl65) and p(DMAPMA65-b-AEMA76) were selected to interact with sulfated BCNC. The 2 wt% of amines or amine polymers in deionized water were dropwise added to the BCNC suspension, and the mixture pH was adjusted to 1.5 with hydrochloric acid. The mixture was stirred at ambient temperature for 6 h. After that the mixture was washed with deionized water by centrifugation method several times until the neutral pH mixture was obtained. Then the cationic BCNC were re-dispersed in deionized water by using vortex mixer.

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Cell viability/cytotoxicity of BCNC. In vitro cytotoxicity test was carried out using MTT assay in HeLa (human cervical carcinoma) cell lines as described in previous work55 and a typical procedure is as follows: Cells, at the density of 10,000 cells/well, were seeded in a 96well plate and cultured in a 100 µL of growth medium consisting Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and antibiotics (penicillin 100 U/mL and streptomycin 100 µg/mL) at 37 °C, 5% CO2. After 24 h, culture media were replaced with DMEM media containing different concentrations of BCNC suspensions. The cells were further incubated for 24 h under the same conditions. Then, 10 µL of sterile filtered MTT stock solution in PBS (5 mg/mL) was added to each well, reaching a final MTT concentration of 0.45 mg/mL. After 4 h, unreacted dye was removed and 100 µL of solubilizing solution (mixture of 2propanol and dimethylsulfoxide in 1:1 volume ratio) was added to each well to dissolve formazan crystals, and the absorbance was measured at 570 nm (GENios, Tecan). The relative cell viability (%) with respect to the control was calculated. All experiments were conducted in triplicate, and average values were reported. Preparation of cationic BCNC-siRNA complexes. Control-siRNA (25 µg) was diluted in 250 µL of Opti-MEM media (OMEM) and complexed with cationic BCNC samples (in OMEM media) at a weight/weight (w/w) ratio of 100. The mixture was incubated at room temperature (23 °C) for 30 min. The siRNA binding ability of the cationic modified BCNC was studied by agarose gel retardation assay (agarose gel electrophoresis). The complexes were loaded in 1% agarose gel containing 1µg/mL ethidium bromide in Tris Acetate/EDTA (TAE) buffer. The gel was run for 45 minutes at 130 V. Then, the siRNA bands were analyzed by an UV transilluminator and pictured by a digital camera. Results and discussion

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Bacterial cellulose (BC) production. For the BC production after incubated for 7 days under static conditions, all three strains of K. xylinus formed smooth and thick BC pellicles at the surface of the culture medium. The optical images and FE-SEM images of resulting BC pellicles were depicted in Figure 1. The micro-structure of the pellicles observed by the FE-SEM revealed that there was no apparent difference in the appearance, and their microfibrils were densely interwoven structures. In this study, the simple culture medium containing only glucose as a carbon source, and yeast extract as a protein and mineral source, was utilized for bacterial growth and bacterial cellulose production. With this medium, all bacterial strains could grow and produce BC in good amounts, which the BC production yields of KX, K1, and K9 strains were 1.8, 1.5, and 1.6 g/L, respectively. It suggested that the bacterial cellulose, a renewable resource of high purity celluloses, can be simply and effectively produced by using these three bacterial strains with the simple fermentative conditions.

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Figure 1. Optical images (left panels) and FE-SEM images viewed with 20,000x magnification (right panels), of BC pellicles produced by three strains of K. xylinus; (A and D) KX, (B and E) K1, and (C and F) K9.

Bacterial cellulose nanocrystals (BCNC) production and characterizations. As shown in Scheme 1, bacterial cellulose nanocrystals (BCNC) were isolated from bacterial cellulose (BC) pellicles by acid hydrolysis to degrade the amorphous region of the microfibrils and then dispersed using sonication. The BCNC yields were dependent of the type of acid used (Table 1), the highest yields were obtained from the hydrochloric acid (H) hydrolysis (around 85%), followed by the mixture of hydrochloric and sulfuric acid (HS) hydrolysis (around 82%), and the lowest ones were from the sulfuric acid (S) hydrolysis (around 80%). The reduction of the BCNC production yields when using the sulfuric acid possibly resulted from its powerful hydrolytic action which can rapidly hydrolyze the less ordered regions of BC chains to separate

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the crystalline domains. The resulted crystalline parts are continuedly reduced in sizes as the surface chains are hydrolyzed and broken into soluble oligo- and mono-saccharides.

Scheme 1. Schematic illustration of the BCNC production from BC by the acid hydrolysis with different types of acid, and the cationic surface modification of sulfated BCNC by the physical adsorption technique with amines and amine-containing polymers (methacrylamide polymers)

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Table 1. Production yield, and crystallinity index (CI) and crystallite sizes (CS) determined from the XRD patterns, of BC pellicles and BCNC produced from three BC sources with different acid hydrolysis conditions. Yield (%)

CI (%)

CS (nm)

-

81

6.6

KX-H

84.4

89

6.5

KX-S

78.6

85

6.3

KX-HS

80.5

87

6.4

-

81

6.7

K1-H

85.1

87

6.6

K1-S

80.2

86

6.3

K1-HS

81.7

88

6.5

-

80

6.8

K9-H

85.6

89

6.7

K9-S

81.5

87

6.3

K9-HS

82.4

89

6.6

Sample KX pellicle

K1 pellicle

K9 pellicle

According to the strong acid hydrolytic activity of sulfuric acid, the BCNC prepared by H2SO4 hydrolysis had the smallest hydrodynamic sizes which were 231.3, 296.5, and 186.9 nm for KXS, K1-H, and K9-HS, respectively (Figure 2A). In addition to the chain scission, the sulfuric acid also has ability to esterify the hydroxyl (OH) groups of cellulose with the sulfate groups (OSO3-) to yield acid half-ester or the so called cellulose sulfate (sulfated cellulose), resulting in the negatively charged surface nanocrystals which can provide the anionic stabilization via the repulsion forces of electrical double layers. Consequently, it helps to prevent the aggregation of nanocrystals driven by hydrogen bonding, then the stable well-dispersed nanocrystal suspension

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can be obtained.18 Therefore, the H2SO4 hydrolyzed BCNC were modified with sulfate groups on their surface and became negatively sulfated BCNC, resulting in the highly negative zeta potential values of -30.9, -32.4, and -31.5 mV for KX-S, K1-S, and K9-S, respectively (Figure 2B). In contrast, the hydrochloric acid has milder hydrolytic action and generally generates cellulose crystallites with native crystalline structures which are free of sulfate groups, resulting in the production of neutral nanocrystals.11 For that reason, the hydrodynamic sizes of the HCl hydrolyzed BCNC were larger; 592.4, 537.5, and 290.1 nm for KX-H, K1-H, and K9-H, respectively, and their zeta potential values were little negative about -11 mV. The occurrence of weak negative charges of the BCNC prepared by HCl hydrolysis could be due to oxidation of the cellulose by oxygen species during bacterial cellulose hydrolysis and purification, causing the negative functional groups such as carboxylic groups on the surface of nanocrystals.58 Although the low surface charges of HCl hydrolyzed BCNC limit their dispersion, they can be useful for applications requiring low charged and/or non-sulfated nanocrystal which will be more difficult and complicated to obtain if they are prepared by H2SO4 hydrolysis adding with a desulfation step. Interestingly, the BCNC prepared by the mixture of HCl and H2SO4 hydrolysis had the particle sizes and zeta potential values in between those prepared by the single acid. The hydrodynamic sizes of KX-HS, K1-HS, and K9-HS were 306.8, 370.5, and 205.2 nm, respectively, and their zeta potential values were around -22 mV, indicating that the hydrolytic action on the starting BC was influenced synergistically by both acids, and the resulting BCNC were esterified with sulfate groups by the sulfuric acid in some extent.

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Figure 2. (A) Hydrodynamic sizes and (B) zeta potential, of BCNC prepared from three BC sources by acid hydrolysis using different types of acid; HCl (H), H2SO4 (S), and combination of HCl and H2SO4 (HS). The FT-IR spectra of BC pellicles and acid hydrolyzed BCNC from all three BC sources exhibited similar cellulose characteristics, thus the FT-IR spectra of K9 source were selected to be representatives as shown in Figure 3. The FT-IR spectra showed typical cellulose vibration bands, such as the strong and broad absorption band at 3550-3200 cm−1 indicated the alcohol OH stretching vibrations (intermolecular bond), including other characteristic peaks: 3350 cm−1 for O-H bond stretching, 2886 cm−1 for C-H bond stretching, 1427 cm−1 for C-H bond symmetric angular deformation, 1370 cm−1 for C-H bond symmetric angular deformation, 1164 cm−1 for CO-C glycoside bond asymmetrical stretching, 1110 cm−1 and 1060 cm−1 for C-OH bond stretching in secondary and primary alcohols, respectively, and 897 cm−1 for C-H bond angular deformation.9,

23

Unfortunately, there was no detectable peak of 807 cm−1 band for C-S bond

symmetric vibration of C-O-SO3- groups, obtaining from esterification of sulfuric acid in the hydrolysis reaction, because the amount of the attached sulfate groups was so small.

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Figure 3. FT-IR spectra of BC and BCNC prepared by acid hydrolysis using HCl (H), H2SO4 (S), and combination of HCl and H2SO4 (HS) from the K9 source. The XRD patterns of BC pellicles and acid hydrolyzed BCNC were shown in Figure 4. All BC pellicles illustrated three 2θ diffraction peaks at 14.5°, 16.7° and 22.7°, while all of resultant BCNC samples showed the peaks at 14.7°, 16.9° and 22.9°, which are usually attributed to the (100), (010), (200) crystallographic planes of cellulose Iα which is the major cellulosic compound in the bacterial cellulose produced by K. xylinus bacterial strains.59

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Figure 4. XRD patterns with three peaks, of BC and BCNC prepared by acid hydrolysis using HCl (H), H2SO4 (S), and combination of HCl and H2SO4 (HS) from three BC sources; (A) KX, (B) K1, and (C) K9.

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Crystallinity is one of the main causes affecting the mechanical properties of materials. Therefore, the XRD patterns were used for determining the crystallinity index (CrI) of native BC and the nanocrystals. The results in Table 1 revealed that the CrI of all acid hydrolyzed BCNC (85-89 %) were greater than the original BC pellicles (around 80%). The increase in crystallinity after acid hydrolysis reaction was the result of a decrease of the amorphous region which is generally more susceptible to acid hydrolysis.60-61 Although the crystallinities of all acid hydrolyzed nanocrystals were nominally the same, when closely comparing the CrI of BCNC prepared by different types of acid, the CrI of H2SO4 hydrolyzed BCNC were the lowest ones, possibly due to the strong hydrolytic action of H2SO4 leading to the digestion of crystalline domains of BC as well.62 In the case of BCNC hydrolyzed by HCl only and mixture of HCl and H2SO4, their crystallinities were comparable with the values of 87-89%. This observation could be explained by the fact that the HCl and the combination of H2SO4 with HCl could provide the suitable hydrolysis conditions which were good to hydrolyze the amorphous region but had little influence on the crystalline region. In addition, the crystallite sizes (CS) of native BC and BCNC were calculated from the XRD spectra by using the Scherrer equation and were shown in Table 1. The BC pellicles exhibited the crystallite sizes of 6.6, 6.7, and 6.8 nm for KX, K1, and K9, respectively, which were larger than their nanocrystal counterparts. The decrease in the crystallite sizes of the acid hydrolyzed BCNC suggested that the acid hydrolysis prone to reduce the crystalline portion of the starting materials.63 Spectacularly, the CS of H2SO4 hydrolyzed BCNC were the smallest (6.3 nm) when comparing to other acid hydrolysis conditions, these results could confirm the strong acid hydrolytic effect of H2SO4 on the studied BC structures.

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The morphology of BCNC suspensions was observed by TEM imaging, it was shown that all BCNC samples displayed the typical and expected rod-like or needle-like nanoparticles, with dimensions of 100–700 nm in length and about 10–30 nm in width. The TEM micrographs of K9-H, K9-S, and K9-HS, representatives of resulting acid hydrolyzed BCNC samples, were illustrated in Figure 5. Interestingly, analyzing the three images, it appeared that the dimensions of H2SO4 hydrolyzed BCNC were more homogeneous, which may be due to the greater electrostatic repulsion of sulfate groups on surface among nanoparticles in aqueous suspension. TGA curves and the corresponding DTG curves of BC and BC nanocrystals obtained by acid

Figure 5. TEM micrographs of the BCNC prepared by different acid hydrolysis; K9-H, K9-S, and K9-HS.

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hydrolysis (Figure. 6) showed that the thermal degradation profiles were similar. There were three main decomposition events in the obtaining weight loss profiles. The first main decomposition event occurring at temperatures below 100 °C was corresponded to the evaporation of adsorbed and bound water plus low molecular weight molecules adsorbed on the surface of materials. For all samples, after the initial weight loss, there was a plateau phase continued to the start of the second main decomposition event which occurred in the temperature range of 250-400 °C. The second main decomposition event was correlated with cellulose degradation which composed of many degradation processes including depolymerization, dehydration and decomposition of glycosidic units. At the high temperature range of 450 to 600 °C, it was the third main decomposition event attributed to the oxidation and decomposition of charred residues to form gaseous products of low molecular weight.12, 20, 64

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Figure 6. TGA (left panels) and DTG (right panels) curves, of BC and BCNC prepared by acid hydrolysis using HCl (H), H2SO4 (S), and combination of HCl and H2SO4 (HS) from three BC sources; (A) KX, (B) K1, and (C) K9. ACS Paragon Plus Environment

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As shown in Table 2, the onset temperature, onset T, which is related to the thermal stability of a sample that indicates the temperature of onset of intense thermal degradation (about 5% weight loss), was higher for all BC pellicles from three BC sources (312, 309, and 318 °C for BC pellicles from KX, K1, and K9, respectively) in comparison with the acid hydrolyzed BCNC counterparts. For the BCNC prepared from the three different BC pellicles (KX, K1, and K9), the nanocrystals hydrolyzed by HCl had higher onset temperature (289, 288, and 310 °C) than those from H2SO4 (265, 270, and 257 °C) and from the mixture of HCl and H2SO4 hydrolysis (290, 269, and 288 °C). The nanocrystals containing sulfate groups are expected to have a lower thermal stability, due to the dehydration catalytic effects of sulfate groups which possibly increase the thermal degradation rate, and the decrease in the activation energy which is caused by the sulfate groups that replaced cellulose hydroxyl groups.12, 64 From the DTG curves shown in Figure 6 (right panels), the BC pellicles and the BCNC from all BC sources demonstrated two peaks of thermal degradation behaviors; the first peak at the lower temperature region represented the maximum degradation temperature (TD1) of the second main decomposition event, and the second peak at the higher temperature region represented the maximum degradation temperature (TD2) of the third main decomposition event. The TD1, TD2, and corresponding weight losses (WL1 and WL2) of the BC pellicles and BCNC produced from three BC sources with different acid hydrolysis conditions were listed in Table 2. It was found that the BC pellicles from all three sources had comparative thermal degradation behaviors with the values of TD1, TD2, WL1, and WL2 about 330 °C, 470 °C, 70% and 30%, respectively. All BCNC prepared by HCl hydrolysis showed a good thermal stability close to their starting BC pellicles, which the TD1 values of KX-H, K1-H, and K9-H were 318, 328, 325 °C, respectively, and their WL1 decreased slightly to be about 60%. In contrast, the thermal stability of the BCNC obtained

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from H2SO4 hydrolysis reduced significantly and their thermal degradation range was broader, resulting from the higher heat susceptibility of sulfate modified crystalline regions.62 For the sulfated BCNC, the degradation process in the second main decomposition event was divided into 2 steps: the first step correlated with the degradation of more accessible regions resulting from the attacking sulfate groups, and the second step correlated with the decomposition of the non-sulfated crystalline regions which were more acid hydrolysis resistance.17,

65

The TD1 and

corresponding WL1 values of K1-S, and K9-S were lower to 294 °C, 41% and 275 °C, 31%, respectively. Whereas, the weight loss of the sulfated BCNC in the third decomposition event (WL2) were increased to 48% for K1-S and 58% for K9-S, suggesting that there were more decomposition of carbonaceous residues and small molecules which might be the results of the desulfation and acid catalyzed dehydration of sulfated nanocrystals in the second event.20 However, the TD1 of KX-S changed lightly to 320 °C with 70% weight loss, but no appearance of the TD2 peak, which might be resulted from the well distribution of sulfate groups on the surface of nanocrystals causing the thermal degradation of most of the material occurred at the same time. Interestingly, the thermal degradation profiles of the BCNC prepared by the mixture of HCl and H2SO4 had the characteristics of both acids as they were used individually, the TD1 and WL1 values were close to HCl hydrolysis, while the broaden peaks in the second decomposition event and the TD2 values were similar to H2SO4 hydrolysis. It demonstrated that the BCNC prepared by the acid mixture were partially sulfated nanocrystals, in which the amounts of attacking sulfated groups might not be enough to cause the decrease in the thermal stability but still be sufficient to affect the formation of char residuals which later decomposed at the lower temperature in the third decomposition event.65

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Table 2. Thermal stability properties determined by TGA; onset temperature, degradation temperature of the first decomposition event (TD1), degradation temperature of the second decomposition event (TD2) and corresponding weight losses (WL1 and WL2) of BC pellicles and BCNC produced from three BC sources with different acid hydrolysis conditions. TD1 (oC)

WL1 (%)

TD2 (oC)

WL2 (%)

KX pellicle 312

330

65.50

460

29.40

KX-H

289

318

45.63

411

35.16

KX-S

265

320

70.15

-

-

KX-HS

290

315

52.25

324

16.99

K1 pellicle

309

328

68.10

468

26.70

K1-H

288

316

60.91

454

37.11

K1-S

270

294

40.84

441

48.18

K1-HS

269

315

62.53

407

30.78

K9 pellicle

318

330

67.83

475

28.35

K9-H

310

325

58.64

441

36.84

K9-S

257

276

30.83

415

57.75

K9-HS

288

316

66.25

400

22.43

Sample

Onset T (oC)

Cationic surface modification of BCNC. Surface modification by means of cationic functionalization is a novel and becomes more interesting method to produce sophisticated nanoparticles. Relative to other approaches, cationic functionalization has been investigated to a lesser extent. In this work, the cationic derivative of BCNC was produced by the conversion of the surface sulfate groups of BCNC to amines with physical adsorption via ionic interaction. For drug delivery applications, the cationic functionalized BCNC should allow the attachment of

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therapeutic proteins, vaccines, or chemotherapeutic drugs. The surface aminated BCNC will allow for protein attachment through the amine groups which can be either the covalent attachment through a linker, or by ionic attachment. Owing to the properties of having negatively charged and the smallest-size nanocrystals, the sulfate hydrolyzed BCNC deriving from the BC pellicle of K9 source (K9-S) were selected to demonstrate the ability of functionalization of BCNC with amines and amine-containing polymers. The K9-S surface charge was highly negative, zeta potential value about -35 mV, because of the presence of the surface sulfate groups resulting from the BCNC production by using sulfuric acid hydrolysis. With these surface sulfate groups, the positive charges of amines can bind onto via the noncovalent electrostatic attraction (physical adsorption). Therefore, the cationic surface modification of BCNC was simply performed by the dropwise addition of the aqueous amine solution into the aqueous sulfated BCNC suspension, then the resulting surface modified BCNC were washed several times with DI water and ultracentrifugation to remove unreacted chemicals. After that, the aminated BCNC were redispersed in DI water to obtain 1 wt% suspension which were measured their hydrodynamic sizes and zeta potential values as shown in Table 3. The results revealed that the particle sizes and zeta potential values of all samples were increased, indicating that the sulfated BCNC were ionically attached with all introduced amines and amine containing polymers. In the case of the surface modification with amines, the zeta potential values of the ethylenediamine modified BCNC (K9-ED), N,Ndimethylethylenediamine modified BCNC (K9-DM),

3-morpholinopropylamine modified

BCNC (K9-MP), and 1-(2-aminoethyl)-piperazine modified BCNC (K9-AP) were around -13, 17, -22, and -15 mV, respectively, which were still negative values. It suggested that only some surface sulfate groups on the BCNC were interacted with the small molecules of amines, and the

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amounts of remaining negative surface charges were greater than the occupied positive ones, resulting in the net negative charges on nanocrystals surface. In contrast, the cationic modified BCNC were successfully developed by the surface modification with amine-containing polymers (methacrylamide polymers in this study), the zeta potential values drastically increased to about +31 and +34 mV for the p(DMAPMA.HCl65) modified BCNC (K9-pD), and p(DMAPMA65-bAEMA76) modified BCNC (K9-pDA), respectively. The results agreed with the notable increase in their sizes (215.7 nm for K9-pD, and 221.6 nm for K9-pDA). It could be explained by the fact that the polymers are macromolecules having numerous amine groups (3° amines in pD, and 1° and 3° amines in pDA) which can interact more with surface sulfate groups on BCNC surface, as well as their long-chain structures can cover more surface area of the nanocrystals, as a result the resultant cationic modified BCNC were larger in size and had net positive charges on their particle surface. Table 3. Hydrodynamic sizes and zeta potential of sulfated BCNC (unmodified) and cationic modified BCNC. Sample

Hydrodynamic size (nm)

Zeta potential (mV)

K9-S

186.9 ± 11.2

-31.5 ± 2.2

K9-ED

193.2 ± 3.5

-12.8 ± 0.7

K9-DM

197.7 ± 6.4

-17.4 ± 1.2

K9-MP

201.4 ± 5.6

-22.1 ± 1.3

K9-AP

203.3 ± 8.4

-15.4 ± 0.5

K9-pD

215.7 ± 6.2

+31.4 ± 1.1

K9-pDA

221.6 ± 7.1

+33.7 ± 0.8

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FT-IR analysis was used to assess the changes of functional groups after the surface modification, and the FT-IR spectra of freeze-dried cationic modified BCNC comparing to the unmodified BCNC (K9-S) were illustrated in Figure 7. All modified BCNC samples showed the characteristic peaks of BCNC. As compared to the unmodified BCNC, the higher intensities of the absorption band at 3000-2850 cm−1 and 1427 cm−1 corresponding to the C-H stretching vibration and the C-H asymmetric angular deformation, respectively, of alkane groups were detected, indicating the achieving surface modification of nanocrystals with amines and polymers which each consists of alkane chains. However, it was difficult to observe the characteristic absorption bands of amine functional groups due to the stretching vibrations of NH stretch at 3350-3300 cm−1 and C-N stretch at 1230-1030 cm−1 are generally weak, and in this study, they were hindered by the strong absorption bands of O-H stretch and C-O stretch, respectively. Fortunately, the absorption bands of the N-H wagging of amines were detectable as a small peak at 860 cm−1 in the spectra of K9-AP, K9-pD, and K9-pDA, presumably resulting from the greater amounts of N-H bonds in these samples. For the K9-MP sample, the intensity of the absorption peak at 1110 cm−1 was higher, presenting the C-O stretching vibrations of the ether group in the morpholine ring of MP. In the cases of the BCNC modified with methacrylamide polymers (K9-pD and K9-pDA), the new absorption peaks at 1650 and 1530 cm-1, attributed to C=O stretching and N-H deformation vibrations in the amide groups, respectively, were observed.

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Figure 7. FTIR spectra of unmodified BCNC (K9-S) and cationic surface modified BCNC with amines (K9-ED, K9-DM, K9-MP, and K9-AP) and with amine-containing polymers (K9-pD, and K9-pDA).

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Cell viability/cytotoxicity of BCNC. Since the cationic modified nanoparticles have been extensively used in biomedical applications, the essential characteristic of developing biomaterials that needs to be evaluated is biocompatibility. The in vitro cytotoxicity is one of important aspects of the biocompatibility testing often used in the biomaterial screening. The cytotoxicity effect of the cationic materials was found to be due to their electrostatic interaction with both extracellular and intracellular membranes and also with proteins in the intracellular space, consequently, the biological pathways were disrupted causing cell apoptosis.64-67 In the evaluation of cell viability changes, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is a widely used method. The MTT is a colorimetric assay which measures amounts of purple formazan produced by viable cells, to determine detrimental intracellular effects of testing substances on mitochondria and metabolic activity.66 Thus, the MTT assay in HeLa cells was performed to study the effects of the cationic modified BCNC on cellular metabolic activity for the cytotoxicity evaluation. The cell viability profile of HeLa cells as a response to increasing concentrations (0.1, 1, and 10 mg/mL) of unmodified and cationic modified BCNC after 24 h exposure was shown in Figure 8A. The cell viability of all samples decreased in relation to the increase of the concentrations. The sulfated BCNC (K9-S) demonstrated the low cytotoxicity effect even at the highest concentration of 10 mg/mL, the cell viability was still high about 73%. The similar result was found in the MP modified BCNC sample (K9-MP), the cell viability was 64% at 10 mg/mL concentration. Due to the presence of the morpholine residues in MP, it has higher hydrophilicity and hence providing a good biocompatibility.67-68 For the rest samples of the BCNC modified with amines, namely K9-ED, K9-DM, and K9-AP, their cell viabilities slightly decreased to around 90% at 0.1 mg/mL, and significantly decreased to around 40% at 10 mg/mL. In the cases

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of the BCNC modified with cationic polymers, the K9-pD and K9-pDA were notably more toxic at higher concentration. At 0.1 mg/mL of K9-pD and K9-pDA, respectively, the cell viabilities were 85% and 84%, and they dropped to 33% and 23% as the concentration increased to 10 mg/mL. The higher cytotoxicity could be resulted from the highly positively surface charged of the nanocrystals which could more electrostatically interact with negative charges of cell membrane causing the higher cell membrane destabilization and disruption, as a result, the cell endocytosis and cell death increased.69-70 Cationic modified BCNC-siRNA complexation. The complexation of cationic modified BCNC with siRNA is based on the interactions between positively charged BCNC and negatively charged siRNA. To confirm that the obtained nanocrystals interacted with siRNA to form complexes, agarose gel electrophoresis retardation assays were performed after mixing siRNA with each of the cationic modified BCNC, as well as unmodified BCNC in a weight ratio of 100, compared to the naked siRNA as a negative control and the polyethylenimine (PEI)siRNA complex as a positive control. The results were illustrated in Figure 8B. The most-left lane was the DNA ladder used for indicating the performance of gel running. In the case of the cationic modified BCNC with amines, all samples displayed free siRNA bands which suggested that the BCNC did not fully complexed with siRNA at this weight ratio used, due to the electrostatic repulsion effect of their net negative charges which could prevent the siRNA from interaction with positively charged regions on the nanocrystals. However, at the same weight ratio, the modified BCNC with cationic polymers showed no free siRNA bands, indicating the full complexation of siRNA which was the result of the highly positive-charged nanocrystals that can effectively neutralize negatively charged siRNA. Interestingly, the bright bands at the wells and long mobilized bands were observed in the unmodified and amine-modified BCNC samples,

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possibly resulting from the electrostatic interaction of negatively charged sulfate groups on the nanocrystals with positively charged amine groups from the ethidium bromide. Conclusion

Figure 8. (A) Cytotoxicity of unmodified and cationic modified BCNC with different concentrations in HeLa cells, after 24-h incubation with cell density of 10,000 cells/well. Data are expressed as percentage of control (nontreated cells 100%, viability) using the mean values and standard deviations from triplicate experiments. (B) Agarose gel electrophoresis retardation assay of unmodified/cationic modified BCNC-siRNA complexes.

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In this work, BC was successfully produced by three different bacterial strains of K. xylinus with a simple fermentative medium containing glucose as a carbon source under static condition. The obtained BC pellicles were then used to produce the BCNC by acid hydrolysis with various acid conditions, namely HCl, H2SO4, and mixture of HCl and H2SO4. The acid conditions and the BC sources profoundly influenced the production yields and the physical properties of nanocrystals including hydrodynamic size, zeta potential, crystallinity index, crystallite size, and thermal stability behavior. Consequently, the BCNC with distinctive characteristics can be simply produced and optimized for designed applications by selecting the proper BC sources and hydrolysis conditions. For example, to demonstrate the potential in biomedical application as a nanocarrier, the sulfated BCNC (K9-S) sample obtained by the H2SO4 hydrolysis of the BC pellicles from K9 source and showed suitable characteristics (hydrodynamic size = 187 nm, zeta potential = -31.5 mV, and CrI = 87%) was selected for the study of the cationic surface modification by physical adsorption with amines and amine-containing polymers. The cationic modified BCNC with high positive surface charges (zeta potential up to +33.7 mV) could be achieved by the uncomplicated electrostatic interaction with methacrylamide polymers, these cationic modified BCNC were similar to the previously reported cationic CNC prepared by the complicated grafting cationic polymers from the CNC surfaces (zeta potential about +40 mV).41 The MTT assay in HeLa cells results revealed that the cytotoxicity of all rod-like cationic modified BCNC was low, the cell viabilities were greater than 80% at the high concentration of 0.1 mg/mL, and they were less toxic than the polyamidoamine-functionalized multi-walled carbon nanotubes which their cell viabilities were less than 30% at the same concentration tested with the same cell lines.38 In addition, the cationic modified BCNC with cationic polymers were fully complexed with siRNA at weight ratio of 100, indicating their potential as nanocarriers for

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nucleic acid delivery; however, the cellular uptake and transfection efficacy demand further investigation to confirm this ability. Therefore, the current study may provide useful information about the production of rod-like BCNC, promising biocompatible biomaterials, which can be simply produced from the BC then surface modified to render the properties of nanocrystals according to the desired applications. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] Phone: +17804921736 *E-mail: [email protected] Phone: +6622184134 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the Natural Sciences and Engineering Council of Canada (NSERC) and by the Petroleum and Petrochemical College, Chulalongkorn University, and funded by the Doctoral Degree Chulalongkorn University 100th Year Birthday Anniversary Scholarship and the Ratchadapisek Sompoch Endowment Fund (2016), Chulalongkorn University (CU-59-026-AM). REFERENCES 1. Qiu, X.; Hu, S., “Smart” Materials Based on Cellulose: A Review of the Preparations, Properties, and Applications. Materials 2013, 6 (3), 738. 2. Nechyporchuk, O.; Belgacem, M. N.; Bras, J., Production of Cellulose Nanofibrils: A Review of Recent Advances. Ind. Crops Prod. 2016, 93 (25 December 2016), 2-25.

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