Cite This: ACS Appl. Nano Mater. 2018, 1,

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1 This article is made available for a limited time sponsored by ACS under the ACS Free to Read License, which permits copying and redistribution of the article for non-commercial scholarly purposes. Cite This: Downloaded via on November 2, 2018 at 10:48:50 (UTC). See for options on how to legitimately share published articles. Bacterial Cellulose Nanocrystals (BCNC) Preparation and Characterization from Three Bacterial Cellulose Sources and Development of Functionalized BCNCs as Nucleic Acid Delivery Systems Pratyawadee Singhsa,, Ravin Narain,*, and 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, Alberta T6G 2G6, Canada ABSTRACT: Bacterial cellulose (BC) is an excellent renewable resource of high-purity cellulose that can be used as original fiber forms or isolated nanocrystalline forms, known as bacterial cellulose nanocrystals (BCNCs), which have gained more attention in the development of highly biocompatible biomaterials. In this work, BCNC production was studied with regard to the influences 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 a mixture of both acids) on the production yield, morphology, and physicochemical properties of the resulting BCNC. BCNC production with these variable factors provided the distinctive characteristics of rodlike nanocrystals, which can be useful for various applications. For demonstration of the biomedical application as nucleic acid delivery systems, cationic BCNCs were developed by 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 BCNCs were evaluated for their complexation ability with sirna and cytotoxicity in HeLa cells. All unmodified and cationic-modified BCNC samples exhibited low toxicity at a concentration of 0.1 mg/ml, which assured their good biocompatibility, and the cationic-modified BCNCs with methacrylamide polymers were fully complexed with sirna. Therefore, this research suggested that a BCNC with the desired properties can be produced by selecting the proper BC sources and acid conditions; also cationic functionalized BCNCs, which revealed their potential as nucleic acid nanocarriers, were easily prepared by simple cationic surface modification. KEYWORDS: bacterial cellulose, bacterial cellulose nanocrystals, acid hydrolysis, crystallinity, cationic surface modification, cytotoxicity, nucleic acid delivery INTRODUCTION Cellulose is a highly available natural biopolymer that has become a good candidate in the development of biomaterials with the 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 because of its higher purity, not containing other kinds of biopolymers, i.e., hemicellulose, pectin, and lignin, which are generally found in plant-derived cellulose. 2 BC is synthesized by various bacterial species; the most effective cellulose-producing species that has often been used as a model organism in BC production studies is Komagataeibacter xylinus (formerly Gluconacetobacter xylinus and Acetobacter xylinum). 3 BC has a distinct ribbon-like 3D network structure (around 100 nm in diameter and around 100 μm in length) that is composed of an extremely fine cellulose fiber, microfibrils of 2 4 nm 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 a BC that 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 (CNCs), or cellulose nanowhiskers, because of its unique characteristics of high purity and high crystallinity Bacterial cellulose nanocrystals (BCNCs) are rodlike nanocrystals obtained from the isolation or extraction of crystalline regions of a BC. The geometrical dimensions of the produced Received: October 29, 2017 Accepted: December 6, 2017 Published: December 6, American Chemical Society 209

2 BCNC are about nm in diameter and nm in length depending on the sources of BC and the isolation methods. 14,15 The main process for the isolation of BCNC from BC fibers is based on acid hydrolysis. Amorphous or disordered sections of cellulose are better hydrolyzed by hydrogen ions, enhancing hydrolytic cleavage of the glycosidic bonds, which releases individual crystallites of the remaining intact crystalline regions that have a higher acid resistance. 16 Several inorganic acids, i.e., hydrochloric (HCl), sulfuric (H 2 SO 4 ), phosphoric, and hydrobromic acids, have been used in acid hydrolysis of cellulose; however, HCl and H 2 SO 4 are more regularly used. 10 The HCl hydrolysis provides nanocrystals with low-density charges on their surface, resulting in a limitation of the nanocrystal dispersibility and an increase in the flocculation of their aqueous suspensions. 17 H 2 SO 4 hydrolysis generates highly negatively charged nanocrystals by sulfonation of the surface hydroxyl groups of cellulose; consequently, highly stable colloidal suspensions can be obtained. 18,19 However, the introduction of surface sulfate groups can cause a decrease in the thermal stability of the resultant nanocrystals. 20,21 Interestingly, the combination of both HCl and H 2 SO 4 acids could tune the surface charge density and improve the thermal stability of the nanocrystals. 22,23 According to appealing intrinsic characteristics of CNCs 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 Furthermore, CNCs 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 In addition, it is a significant advantage to use rodlike CNCs as drug carriers because such nanoparticles were found to be cell internalized faster than spherical nanoparticles. 35,36 Because of their outstanding properties, the rodlike CNCs were also expected to replace carbon nanotubes, which are well-known for their excellent penetrability and encapsulation efficiency for drug and gene delivery but poor biocompatibility Particularly, the surface of CNCs has an abundance of hydroxyl groups, which can be easily modified to other functional groups for the noncovalent and covalent attachment of biomolecules. As a consequence, facile nanocarriers for targeted drug-delivery applications can be developed by the surface modification of CNCs. For the purpose of potential biomedical applications, cationic-modified CNCs have recently been attractive for their ability to interact with anionic active substances for drug delivery 37,40 and also negatively charged nucleic acids for gene delivery. 37,41 The cationic surface modification of CNCs can be accomplished by attaching small molecules or polymers via noncovalent or covalent interactions. There are a few reports about cationization of CNCs, most of which have been performed by chemical modification, including nucleophilic addition of the activated hydroxyl groups on cellulose to the epoxy moiety of epoxypropyltrimethylammonium chloride 42 and cationic polymer grafting by a surface-initiated singleelectron-transfer living radical polymerization method. 41 Although highly effective, chemical modification techniques usually involve tedious and delicate reaction/polymerization procedures and are inconvenient. In the physical modification, CNCs 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 CNCs. In gene therapeutic applications, RNA interference (RNAi) has emerged as an attractive route in which the affected gene can be silenced by delivering small interfering RNA (sirna) to the diseased cells. 47 However, the delivery of sirna is challenging because of the problems of enzymatic degradation and immunological responses of naked sirna and its negative charge property, which limits its penetration ability through a negatively charged cell membrane. 48 Therefore, nanocarriers are necessary to efficiently deliver sirna to the desired target cells; one of the most studied nanocarriers is a nonviral carrier fabricated from synthetic cationic polymers, for which there has been extensive concern about its high toxicity. 53,54 According to that, new delivery nanocarriers developed from biomaterials have become very interesting. Because of the characteristics of nanosized and rodlike particles with good biocompatibility, CNCs can be good candidates for the development of efficient nanocarriers with a low cytotoxicity effect for sirna delivery (nucleic acid delivery). Thus, it was fascinating to produce BCNCs with various properties that can be further appropriately selected for any desired applications, by using various BC sources (three different BC pellicles) and acid types (hydrochloric acid, sulfuric acid, and a mixture of both acids). The structural characteristics of the resulting BCNCs 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 BCNCs in biomedical applications, functionalized (cationic) BCNCs as nucleic acid delivery systems were developed 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 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, and calcium carbonate (CaCO 3 ) were obtained from Sigma-Aldrich. Hydrochloric acid (HCl; ACS reagent, 37%), sulfuric acid (H 2 SO 4 ), 2-propanol, and dimethyl sulfoxide were purchased from Caledon Laboratory Chemicals. Sodium hydroxide (NaOH), ethylenediamine (EDA), N,N-dimethylethylenediamine (DM), 3-morpholinopropylamine (MP), and 1-(2-aminoethyl)- piperazine (AP) were purchased from Sigma-Aldrich. Two of the methacrylamide polymers, namely, poly[n-[3-(dimethylamino)- propyl]methacrylamide hydrochloride] [p(dmapma HCl 65 ), M n = 13500, M w /M n = 1.21] and poly[n-[3-(dimethylamino)propyl]- methacrylamide hydrochloride-co-(2-aminoethyl)methacrylamide hydrochloride] [p(dmapma 65 -b-aema 76 ), M n = 27500, M w /M n = 1.34], were prepared and characterized, as shown in a previous work. 55 Milli-Q water was used for all of the experiments. BC Production. Each of the K. xylinus strains was cultured on glucose yeast extract (GYE) agar containing 100 g of D-glucose, 10 g of yeast extract, 5 g of peptone, 20 g of CaCO 3, and 25 g/l of agar at 30 C for 3 days. Working cultures were routinely prepared on GYE and stored at 4 C until use. For media use, glucose yeast extract broth (GYB) was selected from the literature and modified for the present 210

3 Scheme 1. Schematic Illustration of BCNC Production from BC by Acid Hydrolysis with Different Types of Acid and Cationic Surface Modification of Sulfated BCNC by Physical Adsorption Techniques with Amines and Amine-Containing Polymers (Methacrylamide Polymers) study. GYB consists of 50 g of glucose and 5 g of yeast extract in a 1 L solution. Before use, all of the media were autoclaved at 121 C for 15 min. The ph was adjusted to 5.0 with HCl or NaOH. For the 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 the six modified GYB media. These seed cultures were incubated for 7 days at 30 C under static conditions. Following growth, bacterial cells were separated from cellulose pellicles in the seed cultures by vigorous 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. BC production was incubated under static conditions 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 h and then washed repeatedly with distilled water until a neutral ph was obtained. BCNC Production. Pretreatment: 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 cellulose paste was treated with each acid solution in a ratio of 1:10 g/ ml with continuous stirring in different conditions as follows: the 4 N HCl solution at reflux temperature for 4 h for HCl hydrolysis (H), the 65% (w/w) H 2 SO 4 solution at 60 C for 2 h for H 2 SO 4 hydrolysis (S), and the 1:1 ratio mixture of a 4 N HCl solution and a 65% (w/w) H 2 SO 4 solution at 60 C for 2 h for the two-acid combination hydrolysis (HS). The hydrolysis reactions were terminated by adding an excess (10-fold) of cold deionized (DI) water. The acidic solution was removed by successive centrifugation at rpm for 10 min until the supernatant became turbid. The sediment was collected and dialyzed (MWCO = ) against DI water until reaching neutral ph. After dialysis, the BCNC content was collected by centrifugation for 20 min at rpm and 10 C. Post-treatment: The BCNC samples were redispersed in DI 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 the medium in liters (g/l), while BCNC production was as the dry weight of freeze-dried BCNC compared to the dry weight of the 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 a constant weight was reached. Fourier Transform Infrared (FT-IR) Spectroscopy. Each BC and BCNC sample was air-dried 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 using the Spectrum software. The f α fraction of the samples was calculated by the following: f α IR = 2.55f 0.32 where f IR of cellulose can be calculated as A α /(A α + A β ) and A α and A β are absorbencies at 750 and 710 cm 1, respectively. X-ray Diffraction (XRD). XRD diagrams of dried BCNC samples were recorded using a Rigaku model SmartLab 4800 diffractometer with a Cu Kα radiation wavelength (λ = 1.54 Å), generated at a voltage of 40 kv and a filament emission of 30 ma. Samples were scanned from the 2θ = 5 to 40 range at a scan speed of 2 /min and a scan step of The crystallinity index (CI) and crystallite size (CS) were calculated based on XRD measurements. CI was calculated from the following equation: I200 Iam CI (%) = 100 I 200 where I 200 is the overall intensity of the peak at 2θ = 22.7 and I am is the intensity of the baseline at 2θ = The CS was determined using the Scherrer equation 57 as following: 211

4 ACS Applied Nano Materials CS = Kλ β cos θ for 45 min at 130 V. Then, the sirna bands were analyzed by an UV transilluminator and pictured by a digital camera. RESULTS AND DISCUSSION BC Production. For BC production after incubation 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 and FE-SEM images of the resulting BC pellicles are depicted in Figure 1. The micro- 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 (fwhm), and θ is the Bragg angle. Electron Microscopy. The dried BC samples were sputter-coated with platinum in preparation for field-emission scanning electron microscopy (FE-SEM) imaging. A Hitachi model S-4800 fieldemission scanning electron microscope was used operating at an accelerated voltage of 5 kv and a magnification of 20. Transmission electron microscopy (TEM) micrographs of BCNC samples were taken in a JEOL 100CX-2 transmission electron microscope at an accelerating voltage of 100 kv. Sample solutions [0.02% (w/v)] were deposited on a carbon-coated grid and allowed to dry, followed by staining with a 2 wt % aqueous uranyl acetate solution. Particle Size and ζ-potential Measurements. The hydrodynamic particle sizes and ζ potentials of the unmodified and cationically modified BCNCs in aqueous solutions (salt free) were measured at 25 C using a ZetaPlus ζ-potential analyzer (Brookhaven Instruments Corp.). The reported value is an average of 10 measurements. All measurements were conducted in triplicate. Thermogravimetric Analysis (TGA). TGA curves were recorded with a TA Instruments model SDT Q600 TGA/DSC system. The samples (ca. 10 mg) were heated from 30 to 600 C with a heating rate of 10 C/min under a nitrogen atmosphere. Derivative TGA (DTG) curves 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 physical adsorption with amines and aminecontaining polymers, as illustrated in Scheme 1. Four structurally different amines (EDA, DM, MP, and 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 % amines or amine polymers in DI water were dropwise added to the BCNC suspension, and the mixture ph was adjusted to 1.5 with HCl. The mixture was stirred at ambient temperature for 6 h. After that, the mixture was washed with DI water by a centrifugation method several times until a neutral ph mixture was obtained. Then the cationic BCNC was redispersed in DI water by using a vortex mixer. Cell Viability/Cytotoxicity of BCNC. In vitro cytotoxicity tests were carried out using MTT assay in HeLa (human cervical carcinoma) cell lines as described in a previous work,55 and a typical procedure is as follows: Cells, at the density of cells/well, were seeded in a 96-well plate and cultured in a 100 μl growth medium consisting of Dulbecco s modified Eagle medium (DMEM) with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 μg/ ml streptomycin) at 37 C and 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 a 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, 100 μl of a solubilizing solution (a mixture of 2-propanol and dimethyl sulfoxide in a 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 the average values were reported. Preparation of Cationic BCNC sirna Complexes. The control sirna (25 μg) was diluted in 250 μl of Opti-MEM (OMEM) media 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 1 μg/ml ethidium bromide in Tris acetate/ethylenediaminetetraacetic acid buffer. The gel was run Figure 1. Optical (left panels) and FE-SEM images viewed with magnification (right panels) of the BC pellicles produced by three strains of K. xylinus: (A and D) KX; (B and E) K1; (C and F) K9. structure of the pellicles observed by 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 BC production. With this medium, all bacterial strains could grow and produce BC in good amounts, of 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 BC, a renewable resource of high-purity cellulose, can be simply and effectively produced by using these three bacterial strains with simple fermentative conditions. BCNC Production and Characterization. As shown in Scheme 1, BCNCs were isolated from the BC pellicles by acid hydrolysis to degrade the amorphous region of the microfibrils and then disperse using sonication. The BCNC yields were dependent on the type of acid used (Table 1); the highest yields were obtained from HCl (H) hydrolysis (around 85%), followed by a mixture of HCl and H2SO4 (HS) hydrolysis (around 82%), and the lowest ones were from H2SO4 (S) hydrolysis (around 80%). The reduction of the BCNC production yields when using H2SO4 possibly resulted from its powerful hydrolytic action, which can rapidly hydrolyze the less ordered regions of BC chains to separate the crystalline 212

5 Table 1. Production Yield, CI, and CS Determined from the XRD Patterns of the BC Pellicles and BCNC Produced from Three BC Sources with Different Acid Hydrolysis Conditions sample yield (%) CI (%) CS (nm) KX pellicle KX-H KX-S KX-HS K1 pellicle K1-H K1-S K1-HS K9 pellicle K9-H K9-S K9-HS domains. The resulting crystalline parts are continually reduced in size as the surface chains are hydrolyzed and broken into soluble oligo- and monosaccharides. According to the strong acid hydrolytic activity of H 2 SO 4, the BCNC prepared by H 2 SO 4 hydrolysis had the smallest hydrodynamic sizes, which were 231.3, 296.5, and nm for KX-S, K1-H, and K9-HS, respectively (Figure 2A). In generates cellulose crystallites with native crystalline structures that 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 nm for KX-H, K1-H, and K9-H, respectively, and their ζ- 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 BC hydrolysis and purification, causing 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 a low-charged and/or nonsulfated nanocrystal, which will be more difficult and complicated to obtain if they are prepared by H 2 SO 4 hydrolysis, adding a desulfation step. Interestingly, the BCNC prepared by a mixture of HCl and H 2 SO 4 hydrolysis had particle sizes and ζ- potential values between those prepared by a single acid. The hydrodynamic sizes of KX-HS, K1-HS, and K9-HS were 306.8, 370.5, and nm, respectively, and their ζ-potential values were around 22 mv, indicating that the hydrolytic action on the starting BC was influenced synergistically by both acids, and the resulting BCNCs were esterified with sulfate groups by H 2 SO 4 to some extent. The FT-IR spectra of BC pellicles and acid-hydrolyzed BCNCs from all three BC sources exhibited similar cellulose characteristics; thus, the FT-IR spectra of the K9 source were selected to be representatives, as shown in Figure 3. The FT-IR Figure 2. (A) Hydrodynamic sizes and (B) ζ potentials of BCNCs prepared from three BC sources by acid hydrolysis using different types of acid: HCl (H), H 2 SO 4 (S), and a combination of HCl and H 2 SO 4 (HS). addition to chain scission, H 2 SO 4 also has the ability to esterify the hydroxyl (OH) groups of cellulose with sulfate groups (OSO 3 ) to yield an acid half-ester or the so-called cellulose sulfate (sulfated cellulose), resulting in negatively charged surface nanocrystals that can provide anionic stabilization via the repulsion forces of electrical double layers. Consequently, this helps to prevent the aggregation of nanocrystals driven by hydrogen bonding, and then the stable well-dispersed nanocrystal suspension can be obtained. 18 Therefore, the H 2 SO 4 - hydrolyzed BCNC was modified with sulfate groups on their surface and became a negatively sulfated BCNC, resulting in the highly negative ζ-potential values of 30.9, 32.4, and 31.5 mv for KX-S, K1-S, and K9-S, respectively (Figure 2B). In contrast, HCl has milder hydrolytic action and generally Figure 3. FT-IR spectra of BC and BCNC prepared by acid hydrolysis using HCl (H), H 2 SO 4 (S), and a combination of HCl and H 2 SO 4 (HS) from the K9 source. spectra showed typical cellulose vibration bands, such as the strong and broad absorption band at cm 1 indicating the alcohol O H 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 asymmetric angular deformation, 1370 cm 1 for C H bond symmetric angular 213

6 deformation, 1164 cm 1 for C O C glycoside bond asymmetric stretching, 1110 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 the 807 cm 1 band for C S bond symmetric vibration of C O SO 3 groups, obtained from esterification of H 2 SO 4 in the hydrolysis reaction, because the amount of the attached sulfate groups was so small. The XRD patterns of BC pellicles and acid-hydrolyzed BCNCs are shown in Figure 4. All BC pellicles illustrated three Figure 4. XRD patterns with three peaks of BC and BCNC prepared by acid hydrolysis using HCl (H), H 2 SO 4 (S), and a combination of HCl and H 2 SO 4 (HS) from three BC sources: (A) KX; (B) K1; (C) K9. 2θ diffraction peaks at 14.5, 16.7, and 22.7, while all of the resulting BCNC samples showed peaks at 14.7, 16.9, and 22.9, which are usually attributed to the (100), (010), and (200) crystallographic planes of cellulose I α, which is the major cellulosic compound in the BC produced by K. xylinus bacterial strains. 59 Crystallinity is one of the main causes affecting the mechanical properties of materials. Therefore, the XRD patterns were used for determining the CIs of native BC and nanocrystals. The results in Table 1 revealed that the CIs of all acid-hydrolyzed BCNCs (85 89%) were greater than those of the original BC pellicles (around 80%). The increase in the crystallinity after an 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, upon a close comparison of the CIs of BCNCs prepared by different types of acid, the CIs of H 2 SO 4 -hydrolyzed BCNCs were the lowest ones, possibly because of the strong hydrolytic action of H 2 SO 4, leading to digestion of the crystalline domains of BC as well. 62 In the case of BCNCs hydrolyzed by HCl only and a mixture of HCl and H 2 SO 4, their crystallinities were comparable with the values of 87 89%. This observation could be explained by the fact that HCl and a combination of H 2 SO 4 and HCl could provide suitable hydrolysis conditions, which were good to hydrolyze the amorphous region but had little influence on the crystalline region. In addition, the CSs of native BC and BCNC were calculated from the XRD spectra by using the Scherrer equation and are shown in Table 1. The BC pellicles exhibited CSs 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 CSs of acid-hydrolyzed BCNCs suggested that acid hydrolysis is prone to reducing the crystalline portion of the starting materials. 63 Spectacularly, the CSs of H 2 SO 4 -hydrolyzed BCNCs were the smallest (6.3 nm) compared to other acid hydrolysis conditions; these results could confirm the strong acid hydrolytic effect of H 2 SO 4 on the studied BC structures. The morphology of BCNC suspensions was observed by TEM imaging. It was shown that all BCNC samples displayed the typical and expected rodlike or needlelike nanoparticles, with dimensions of nm in length and about nm in width. The TEM micrographs of K9-H, K9-S, and K9- HS, representatives of the resulting acid-hydrolyzed BCNC samples, are illustrated in Figure 5. Interestingly, upon analysis of the three images, it appeared that the dimensions of H 2 SO 4 - hydrolyzed BCNCs were more homogeneous, which may be due to the greater electrostatic repulsion of sulfate groups on the surface among nanoparticles in an aqueous suspension. TGA curves and the corresponding DTG curves of BC and BC nanocrystals obtained by acid 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 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, a plateau phase continued to the start of the second main decomposition event, which occurred in the temperature range of C. The second main decomposition event was correlated with cellulose degradation, which was composed of many degradation processes including depolymerization, dehydration, and decomposition of glyco- 214

7 ACS Applied Nano Materials maximum degradation temperature (TD2) of the third main decomposition event. TD1, TD2, and the corresponding weight losses (WL1 and WL2) of the BC pellicles and BCNCs produced from three BC sources with different acid hydrolysis conditions are listed in Table 2. It was found that the BC pellicles from all three sources had comparative thermal degradation behavior with values of TD1, TD2, WL1, and WL2 of about 330 C, 470 C, 70%, and 30%, respectively. All BCNCs prepared by HCl hydrolysis showed a good thermal stability close to their starting BC pellicles, of which the TD1 values of KX-H, K1-H, and K9-H were 318, 328, and 325 C, respectively, and their WL1 value decreased slightly to about 60%. In contrast, the thermal stability of the BCNCs obtained from H2SO4 hydrolysis reduced significantly, and their thermal degradation range was broader, resulting from the higher heat susceptibility of the sulfate-modified crystalline regions.62 For sulfated BCNCs, the degradation process in the second main decomposition event was divided into two steps: the first step correlated with the degradation of more accessible regions, resulting from the attacking sulfate groups, and the second step correlated with decomposition of the nonsulfated crystalline regions, which were more acid hydrolysis resistant.17,65 The TD1 and corresponding WL1 values of K1-S and K9-S were lower at 294 C, 41% and 275 C, 31%, respectively, whereas the weight loss of the sulfated BCNCs in the third decomposition event (WL2) were increased to 48% for K1-S and 58% for K9-S, suggesting that there was more decomposition of carbonaceous residues and small molecules, which might be the result of desulfation and acid-catalyzed dehydration of sulfated nanocrystals in the second event.20 However, the TD1 value of KX-S changed slightly to 320 C with 70% weight loss, but no appearance of the TD2 peak, which might have resulted from the well distribution of sulfate groups on the surface of nanocrystals, causing thermal degradation of most of the material, occurred at the same time. Interestingly, the thermal degradation profiles of BCNCs prepared by a mixture of HCl and H2SO4 had the characteristics of both acids as if they were used individually. The TD1 and WL1 values were close to those of HCl hydrolysis, while the broadened peaks in the second decomposition event and the TD2 values were similar to those of H2SO4 hydrolysis. This demonstrated that the BCNCs prepared by the acid mixture were partially sulfated nanocrystals, in which the amounts of attacking sulfated groups might not be enough to cause a decrease in the thermal stability but might still be sufficient to affect the formation of char residuals, which later decomposed at lower temperature in the third decomposition event.65 Cationic Surface Modification of BCNCs. Surface modification by means of cationic functionalization is a novel and 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 BCNCs was produced by conversion of the surface sulfate groups of BCNCs to amines with physical adsorption via ionic interaction. For drug-delivery applications, the cationic-functionalized BCNCs should allow the attachment of therapeutic proteins, vaccines, or chemotherapeutic drugs. The surface-aminated BCNCs will allow for protein attachment through the amine groups, which can be either covalent attachment through a linker or ionic attachment. Because of the properties of negative charge and the smallestsized nanocrystals, sulfate-hydrolyzed BCNCs derived from the BC pellicles of the K9 source (K9-S) were selected to Figure 5. TEM micrographs of the BCNC prepared by different acid hydrolyses: K9-H, K9-S, and K9-HS. sidic units. At the high-temperature range of C, there was a third main decomposition event attributed to the oxidation and decomposition of charred residues to form gaseous products of low molecular weight.12,20,64 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 BCNCs prepared from the three different BC pellicles (KX, K1, and K9), the nanocrystals hydrolyzed by HCl had higher onset temperatures (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 because of the dehydration catalytic effects of the sulfate groups, which possibly increase the thermal degradation rate and decrease in the activation energy, which is caused by sulfate groups replacing cellulose hydroxyl groups.12,64 From the DTG curves shown in Figure 6 (right panels), the BC pellicles and BCNCs 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 215

8 Figure 6. TGA (left panels) and DTG (right panels) curves of BC and BCNC prepared by acid hydrolysis using HCl (H), H 2 SO 4 (S), and a combination of HCl and H 2 SO 4 (HS) from three BC sources: (A) KX; (B) K1; (C) K9. demonstrate the ability of functionalization of BCNCs with amines and amine-containing polymers. The K9-S surface charge was a highly negative, ζ-potential value of about 35 mv because of the presence of the surface sulfate groups resulting from BCNC production using H 2 SO 4 hydrolysis. With these surface sulfate groups, the positive charges of amines can bind via noncovalent electrostatic attraction (physical adsorption). Therefore, the cationic surface modification of BCNC was simply performed by the dropwise addition of an aqueous amine solution into an aqueous sulfated BCNC suspension, and then the resulting surface-modified BCNCs were washed several times with DI water and ultracentrifuged to remove unreacted chemicals. After that, the aminated BCNCs were redispersed in DI water to obtain a 1 wt % suspension, for which the hydrodynamic sizes and ζ-potential values were measured, as shown in Table 3. The results revealed that the particle sizes and ζ-potential values of all samples were increased, indicating that the sulfated BCNCs were ionically attached with all introduced amines and amine-containing polymers. In the case of the surface modification with amines, the ζ-potential values of the EDA-modified BCNC (K9-ED), DM-modified BCNC (K9-DM), MP-modified BCNC (K9- MP), and AP-modified BCNC (K9-AP) were around 13, 17, 22, and 15 mv, respectively, which were still negative values. This suggested that only some surface sulfate groups on the BCNCs interacted with the small molecules of amines, and the amounts of the remaining negative surface charges were greater than the occupied positive ones, resulting in net negative charges on the nanocrystal surfaces. In contrast, the cationic-modified BCNCs were successfully developed by surface modification with amine-containing polymers (methacrylamide polymers in this study), the ζ-potential values drastically increased to about +31 and +34 mv for the p(dmapma HCl 65 )-modified BCNC (K9-pD) and the p- 216

9 Table 2. Thermal Stability Properties Determined by TGA, Onset Temperature, Degradation Temperature of the First Decomposition Event (T D1 ), Degradation Temperature of the Second Decomposition Event (T D2 ), and Corresponding Weight Losses (WL 1 and WL 2 ) of BC Pellicles and BCNCs Produced from Three BC Sources with Different Acid Hydrolysis Conditions sample onset T ( C) T D1 ( C) WL 1 (%) T D2 ( C) WL 2 (%) KX pellicle KX-H KX-S KX-HS K1 pellicle K1-H K1-S K1-HS K9 pellicle K9-H K9-S K9-HS Table 3. Hydrodynamic Sizes and ζ Potentials of Sulfated BCNCs (Unmodified) and Cationic-Modified BCNCs sample hydrodynamic size (nm) ζ potential (mv) K9-S ± ± 2.2 K9-ED ± ± 0.7 K9-DM ± ± 1.2 K9-MP ± ± 1.3 K9-AP ± ± 0.5 K9-pD ± ± 1.1 K9-pDA ± ± 0.8 (DMAPMA 65 -b-aema 76 )-modified BCNC (K9-pDA), respectively. The results agreed with the notable increase in their sizes (215.7 nm for K9-pD and nm for K9-pDA). This 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) that can interact more with surface sulfate groups on the BCNC surface, and their long-chain structures can cover more surface area of the nanocrystals; as a result, the resulting cationic-modified BCNCs were larger in size and had net positive charges on their particle surfaces. FT-IR analysis was used to assess the changes of the functional groups after surface modification, and the FT-IR spectra of freeze-dried cationic-modified BCNCs compared to the unmodified BCNCs (K9-S) are illustrated in Figure 7. All modified BCNC samples showed the characteristic peaks of BCNCs. Compared to the unmodified BCNCs, higher intensities of the absorption bands at and 1427 cm 1 corresponding to the C H stretching vibration and C H asymmetric angular deformation, respectively, of alkane groups were detected, indicating the achievement of surface modification of nanocrystals with amines and polymers, each consisting of alkane chains. However, it was difficult to observe the characteristic absorption bands of amine functional groups because the stretching vibrations of the N H stretch at cm 1 and the C N stretch at cm 1 are generally weak, and in this study, they were hindered by strong absorption bands of O H and C O stretching, respectively. Fortunately, the absorption bands of the N H wagging of amines were detectable as a small peak at 860 cm 1 in the Figure 7. FT-IR spectra of unmodified BCNC (K9-S) and cationic surface-modified BCNCs with amines (K9-ED, K9-DM, K9-MP, and K9-AP) and with amine-containing polymers (K9-pD and K9-pDA). 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 case of 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. Cell Viability/Cytotoxicity of BCNC. Because the cationicmodified 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 the 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 217

10 intracellular space; consequently, the biological pathways were disrupted, causing cell apoptosis In the evaluation of the cell viability changes, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a widely used method. MTT is a colorimetric assay that measures the amounts of purple formazan produced by viable cells to determine the detrimental intracellular effects of testing substances on mitochondria and metabolic activity. 66 Thus, MTT assay in HeLa cells was performed to study the effects of the cationicmodified BCNCs on the 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 BCNCs after 24 h of exposure is shown in Figure 8A. The cell viability of all Figure 8. (A) Cytotoxicity of unmodified and cationic-modified BCNCs with different concentrations in HeLa cells, after 24 h of incubation with a cell density of cells/well. Data are expressed as a percentage of the 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. 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, and the cell viability was still high at about 73%. A similar result was found in the MP-modified BCNC sample (K9-MP), and the cell viability was 64% at 10 mg/ml concentration. Because of the presence of morpholine residues in MP, it has a higher hydrophilicity, hence providing good biocompatibility. 67,68 For the rest of the samples of BCNCs 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 of the BCNCs modified with cationic polymers, 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 might have resulted from the high positive surface charge of the nanocrystals, which could more electrostatically interact with the negative charges of the cell membrane, causing 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 BCNCs, as well as unmodified BCNC in a weight ratio of 100, compared to the naked sirna as a negative control and the polyethylenimine sirna complex as a positive control. The results are illustrated in Figure 8B. The leftmost lane is the DNA ladder used for indicating the performance of gel running. In the case of cationic-modified BCNCs with amines, all samples displayed free sirna bands, which suggested that the BCNC did not fully complex with sirna at the weight ratio used because of the electrostatic repulsion effect of their net negative charges, which could prevent the sirna from interacting with positively charged regions on the nanocrystals. However, at the same weight ratio, the modified BCNCs with cationic polymers showed no free sirna bands, indicating full complexation of sirna, which was the result of highly positively 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, possibly resulting from the electrostatic interaction of negatively charged sulfate groups on the nanocrystals with positively charged amine groups from ethidium bromide. CONCLUSION In this work, BC was successfully produced by three different bacterial strains of K. xylinus with a simple fermentative medium containing glucose as the carbon source under static conditions. The obtained BC pellicles were then used to produce BCNCs by acid hydrolysis with various acid conditions, namely, HCl, H 2 SO 4, and a mixture of HCl and H 2 SO 4. The acid conditions and BC sources profoundly influenced the production yields and physical properties of nanocrystals including the hydrodynamic size, ζ potential, CI, CS, and thermal stability behavior. Consequently, the BCNCs 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 its potential for biomedical application as a nanocarrier, the sulfated BCNC (K9-S) sample obtained by H 2 SO 4 hydrolysis of the BC pellicles from the K9 source and showing suitable characteristics (hydrodynamic size = 187 nm, ζ potential = 31.5 mv, and CI = 87%) was selected for the study of cationic surface modification by physical adsorption with amines and amine-containing polymers. Cationic-modified BCNCs with 218

11 high positive surface charges (ζ potential of up to mv) could be achieved by the uncomplicated electrostatic interaction with methacrylamide polymers; these cationicmodified BCNCs were similar to the previously reported cationic CNC prepared by complicated grafting cationic polymers from the CNC surfaces (ζ potential of about +40 mv). 41 The MTT assay in HeLa cells results revealed that the cytotoxicity of all rodlike cationic-modified BCNCs was low, the cell viabilities were greater than 80% at a high concentration of 0.1 mg/ml, and they were less toxic than the polyamidoamine-functionalized multiwalled carbon nanotubes, for which the cell viabilities were less than 30% at the same concentration as that tested with the same cell lines. 38 In addition, the cationic-modified BCNCs with cationic polymers were fully complexed with sirna at a weight ratio of 100, indicating their potential as nanocarriers for nucleic acid delivery; however, the cellular uptake and transfection efficacy demand further investigation to confirm this ability. 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