基于入侵植物龙舌兰和蓖麻提取的具有细胞相容性的纳米纤维：一种高结晶性纳米纤维素的可再生的绿色资源

Cytocompatible cellulose nanofibers from invasive plant species Agave americana L. and Ricinus communis L.: a renewable green source of highly crystalline nanocellulose 基于入侵植物龙舌兰和蓖麻提取的具有细胞相容性的纳米纤维：一种高结晶性纳米纤维素的可再生的绿色资源 1 first-author 1 1 2 3 corresp Zaida ORTEGA, zaida.ortega@ulpgc.es Zaida ORTEGA, zaida.ortega@ulpgc.es zaida.ortega@ulpgc.es https://orcid.org/0000-0002-7112-1067 1 1 4 corresp João RODRIGUES, joaor@uma.pt; joaoc@staff.uma.pt João RODRIGUES, joaor@uma.pt; joaoc@staff.uma.pt joaor@uma.pt https://orcid.org/0000-0003-4552-1953 CQM‒Centro de Química da Madeira, MMRG, Universidade da Madeira, Campus Universitário da Penteada, 9020-105Funchal, Portugal CQM‒Centro de Química da Madeira, MMRG, Universidade da Madeira, Campus Universitário da Penteada, 9020-105Funchal, Portugal Faculty of Polytechnics, University of Donja Gorica, Oktoih 1, 81000 Podgorica, Montenegro Faculty of Polytechnics, University of Donja Gorica, Oktoih 1, 81000 Podgorica, Montenegro Departamento de Ingeniería de Procesos, Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Las Palmas, Spain Departamento de Ingeniería de Procesos, Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Las Palmas, Spain School of Materials Science and Engineering/Center for Nano Energy Materials, Northwestern Polytechnical University, Xi'an710072, China School of Materials Science and Engineering/Center for Nano Energy Materials, Northwestern Polytechnical University, Xi'an710072, China In this study, the fibers of invasive species Agave americana L. and Ricinus communis L. were successfully used for the first time as new sources to produce cytocompatible and highly crystalline cellulose nanofibers. Cellulose nanofibers were obtained by two methods, based on either alkaline or acid hydrolysis. The morphology, chemical composition, and crystallinity of the obtained materials were characterized by scanning electron microscopy (SEM) together with energy-dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. The crystallinity indexes (CIs) of the cellulose nanofibers extracted from A. americana and R. communis were very high (94.1% and 92.7%, respectively). Biological studies evaluating the cytotoxic effects of the prepared cellulose nanofibers on human embryonic kidney 293T (HEK293T) cells were also performed. The nanofibers obtained using the two different extraction methods were all shown to be cytocompatible in the concentration range assayed (i.e., 0‍‒‍500 µg/mL). Our results showed that the nanocellulose extracted from A. americana and R. communis fibers has high potential as a new renewable green source of highly crystalline cellulose-based cytocompatible nanomaterials for biomedical applications. 创新点 2 本研究中，首次使用入侵植物龙舌兰和蓖麻纤维来生产具有细胞相容性和高度结晶的纤维素纳米纤维。 目的 2 探索入侵植物龙舌兰和蓖麻作为制备高结晶纤维素纳米纤维的可再生资源的潜力。 方法 2 通过碱性或酸性水解方法来制备纤维素纳米纤维；利用扫描电子显微镜（SEM）结合X射线能谱（EDX）、动态光散射（DLS）、X射线衍射（XRD）以及傅立叶变换红外光谱（FTIR）对获得的材料的表型、化学组成和结晶度进行了分析表征。 结论 2 本研究发现从美国龙舌兰和蓖麻中提取的纤维素纳米纤维的结晶度指数（CI）很高，分别为94.1%和92.7%。通过评估所制备的纤维素纳米纤维对HEK293T细胞的细胞毒性作用，发现两种不同提取方法获得的纳米纤维在所测定的浓度范围内（即0-500 µg/mL）均显示出细胞相容性。研究结果表明，从龙舌兰和蓖麻纤维中提取的纳米纤维素可作为一种新型可再生的绿色资源，制备高结晶纤维素且具有细胞相容性的纳米材料，具有很高的生物医学应用潜力。 Nanofiber Nanocellulose Agave americana L. Ricinus communis L. Crystallinity Invasive species Biomedical application 纳米纤维 纳米纤维素 龙舌兰属 蓖麻属 结晶度 入侵物种 生物医学应用 1 Introduction 1 The increasing number of invasive species worldwide is considered a global environmental and economic problem and a major threat to biodiversity ( Charles and Dukes, 2007 Charles and Dukes, 2007 ). These invasive species are damaging the native fauna and flora of ecosystems, leading to severe threats to local biodiversity, agricultural and forest production, and ultimately to human health ( Mooney and Hobbs, 2000 Mooney and Hobbs, 2000 ; Crowl et al., 2008 Crowl et al., 2008 ). Invasive plant species can be exploited as a cheap and widely available renewable source of cellulosic fibers ( Satyanarayana et al., 2013 Satyanarayana et al., 2013 ; Sun et al., 2020 Sun et al., 2020 ). At present, the exploitation of various non-traditional sources of cellulose (such as annual plants, vegetables, weeds, residues from crops such as wheat/vine stems, corn grain/cobs, sugarcane bagasse, and marine residues) is gaining more importance as a way to reduce the high consumption of natural fibers and to find new strategies for their potential application and valorization ( Aguir and M'Henni, 2006 Aguir and M'Henni, 2006 ; Fiserova et al., 2006 Fiserova et al., 2006 ; Abrantes et al., 2007 Abrantes et al., 2007 ; Khiari et al., 2011 Khiari et al., 2011 ; Ridzuan et al., 2016 Ridzuan et al., 2016 ). For instance, Abrantes et al. (2007) Abrantes et al. (2007) used annually harvested non-wood fibers from Cynara cardunculus L. (cardoon) for paper and paperboard production. Maheswari et al. (2012) Maheswari et al. (2012) extracted cellulose microfibrils from the agricultural residue of coconut palm leaf sheaths. Another research group ( Fiore et al., 2014 Fiore et al., 2014 ) studied the possibility of using an invasive plant, Arundo donax L., as a source of reinforcement fibers in polymer composites. Our team recently explored the use of pure Agave americana L. fibers for reinforcement in polymer composites ( Ortega et al., 2019 Ortega et al., 2019 ). Within the past decade, nanoscale cellulose has demonstrated great potential in the biomedical field, namely, in drug delivery, tissue engineering, cartilage replacements, medical implants, skin wound dressings, bioimaging, and biosensing ( Lin and Dufresne, 2014 Lin and Dufresne, 2014 ; Guise and Fangueiro, 2016 Guise and Fangueiro, 2016 ; Kargarzadeh et al., 2017 Kargarzadeh et al., 2017 ; Mishra et al., 2018 Mishra et al., 2018 ; Xu et al., 2018 Xu et al., 2018 ). Nanocellulose has been exploited as a biomaterial for three-dimensional (3D) printing to design drug delivery devices with the desired drug release profile for personalized therapy ( Yu et al., 2009 Yu et al., 2009 ; Sandler and Preis, 2016 Sandler and Preis, 2016 ). Lately, nanocellulose has been increasingly utilized as 3D scaffolds in liver tissue engineering, adipose tissue engineering, and vascular tissue engineering ( Bacakova et al., 2019 Bacakova et al., 2019 ). Rees et al. (2015) Rees et al. (2015) explored the application of nanocellulose as a bioink for possible use as a wound‍-dressing material. Åhlén et al. (2018) developed contact lenses based on cellulose-nanocrystal-reinforced polyvinyl alcohol that could be used for controlled ocular drug delivery. Bacterial nanocellulose has been applied as a new patch for the repair of congenital heart defects ( Lang et al., 2015 Lang et al., 2015 ). Another interesting use of nanocellulose was reported by Wang et al. (2016) Wang et al. (2016) . They obtained biocomposites of nanofibrillated cellulose for dressing materials in an emerging concept of Cu-containing wound healing dressings. Numerous studies have demonstrated the possibility of obtaining cellulose nanofibers, nanocrystals, or nanofibrils from non-conventional cellulose resources ( Oun and Rhim, 2016 Oun and Rhim, 2016 ; Bajpai, 2017 Bajpai, 2017 ; Chen and Lee, 2018 Chen and Lee, 2018 ; Pereira and Arantes, 2018 Pereira and Arantes, 2018 ). Reddy and Rhim (2014) Reddy and Rhim (2014) demonstrated that paper-mulberry ( Broussonetia kazinoki Siebold) bast pulp could be used as a new, environmentally friendly nanocellulose source. Bettaieb et al. (2015a Bettaieb et al. (2015a , 2015b) 2015b) reported on the preparation of a new source of nanocellulose crystals extracted from Posidonia oceanica leaves and balls. Nanocellulose fibers with a high crystallinity index (CI) of about 70% and improved thermal stability were produced from pinecones (Jack pine: Pinus banksiana Lamb.) using chemical and mechanical treatments ( Rambabu et al., 2016 Rambabu et al., 2016 ). Chen and Lee (2018) Chen and Lee (2018) recently isolated nanocellulose from Panax ginseng , spent tea residue, waste cotton cloth, and old corrugated cardboard, and demonstrated its potential in commercial applications for sustainable development. For the islands of the Macaronesia archipelagos (Azores (Portugal), Madeira (Portugal), Canary Islands (Spain), and Cape Verde) and other regions of the world, the revalorization of invasive plants such as A. americana , A. donax (giant reed), Pennisetum setaceum , and Ricinus communis for the production of biomaterials and composites would constitute an environmental benefit and an attractive economic income that would support eradication campaigns. The plants mentioned above are currently utilized only as a source of cellulose fibers for the textile and automobile industries ( Ridzuan et al., 2016 Ridzuan et al., 2016 ; Motaung et al., 2017 Motaung et al., 2017 ; Trifol et al., 2017 Trifol et al., 2017 ; Vinayaka et al., 2017 Vinayaka et al., 2017 ). To the best of our knowledge, no attempts have been made to use the fibers of these invasive plant species as an inexpensive nanocellulose source for biomedical applications. In the present study, we chose the fast-growing, widely distributed, and highly abundant plants A. americana and R. communis to produce cytocompatible nanoscaled cellulose. The exploration and revalorization of these plants are particularly crucial for the Macaronesia archipelagos island, where they present a severe environmental problem since they occupy abandoned agricultural land and all areas not cared for or that have been victims of the fire. Therefore, the primary objective of the study was to explore the potential of the selected invasive plants as an inexpensive and abundantly available renewable source of natural, highly crystalline cellulose nanofibers. 2 Materials and methods 1 2.1 Materials 2 Two samples of invasive plants, A. americana and R. communis , were collected from the Macaronesia archipelago, specifically on Gran Canaria island (Canary Islands archipelago, Spain); plants were collected from abandoned farmland (global positioning system (GPS) coordinates: 28.079 766, -‍15.455 594) now transformed into the urban ground, and oven-dried for 24 ‍h at 105 °‍C before further use. All reagents were purchased from Sigma Aldrich (Madrid, Spain or Lisbon, Portugal) and were used without further purification. 2.2 Cellulose extraction procedure 2 Cellulose nanofibers were extracted from two different raw material sources, A. americana and R. communis. Before extraction, the raw, dried, and milled plants were processed by refluxing with toluene-ethanol (2:1, volume ratio) for 8 h in a Soxhlet apparatus to remove pigments, waxes, and lipids. The raw materials were then thoroughly washed on a Büchner funnel with ethanol, acetone, and distilled water, and dried at room temperature until a constant weight was achieved. Subsequently, two different methods were used for cellulose extraction, as presented in Fig. 1 Fig. 1 and detailed below. 2.2.1　Extraction method #1, based on an alkali treatment 3 The dewaxed dried samples were treated with 15% (mass fraction) of NaOH solution at 60 °‍C for 1 h under mechanical stirring, using a fiber to liquid ratio of 1:15. After filtration and extensive washing with distilled water on a Büchner funnel, the residue was dried at room temperature until a constant weight was achieved. The alkali-treated fibers were bleached with a mixture of 3% NaOH, 2% H 2 O 2 , and 3% Na 2 SiO 3 (all at mass fraction) solution at 70 °C for 1.5 h under mechanical stirring using a fiber to liquid ratio of 1:60. This treatment was repeated twice. After bleaching, the fibers were thoroughly washed with distilled water until a neutral pH was obtained and then dried overnight until a constant weight was achieved. The prepared A. americana and R. communis fibers were mixed with a 60% (mass fraction, the same below) H 2 SO 4 solution and stirred vigorously at 45 °‍C for 2.5 and 4.5 h, respectively. After that, hydrolysis was immediately quenched by adding 500 mL of cold distilled water to the reaction mixture. The resulting nanocellulose suspension was centrifuged several times (10 000 r/min, 10 min) to separate the nanocellulose from the sulphuric acid solution. The final suspension was continuously dialyzed (dialysis membrane 1000 Da) against distilled water to remove the sulfate ions until a constant, neutral pH was reached. Finally, the obtained nanocellulose suspension was freeze-dried and stored at 8 °‍C for further characterization. 2.2.2　Extraction method #2, based on acid hydrolysis 3 The dewaxed dried samples were pretreated using the Kurschner-Hoffer method ( Béakou et al., 2008 Béakou et al., 2008 ). The resulting sample was refluxed with a mixture of nitric acid and ethyl alcohol (1:4, volume ratio) for 4 h in total. The residue was filtered off and then refluxed again with the alcohol/nitric acid solution. This procedure was repeated three times. Finally, the residue was filtered off and washed with hot distilled water until a neutral pH was achieved. This was followed by drying in an oven at 60 °‍C until a constant weight was obtained. Then, the prepared A. americana fibers were mixed with a 40% H 2 SO 4 solution and stirred vigorously at 60 °‍C for 2 h. R. communis fibers were mixed with a 60% H 2 SO 4 solution and stirred vigorously at 45 °‍C for 2.5 h. The ratio of fibers to the acid solution was 1:40. After that, hydrolysis was immediately quenched by adding 500 mL of cold distilled water to the reaction mixture. Non-reactive sulfate groups were removed from the resulting suspension by centrifugation, separating the nanocellulose from the solution after each washing step. The final suspension was continuously dialyzed (dialysis membrane 1000 Da) against distilled water until a constant neutral pH was reached. Finally, the obtained nanocellulose suspension was freeze-dried and stored at 8 °‍C for further characterization. The details of the extraction conditions for each prepared sample are provided in Table 1 Table 1 . CNF: cellulose nanofiber. Sample name Source Extraction method H 2 SO 4 (%, mass fraction) Temperature (°C) Hydrolysis time (h) CNF AA1 Agave americana Method #1 60 45 2.5 CNF AA2 A. americana Method #2 40 60 2.0 CNF RC1 Ricinus communis Method #1 60 45 4.5 CNF RC2 R. communis Method #2 60 45 2.5 2.3 Characterization 2 2.3.1　Chemical composition of raw materials 3 The chemical composition of the raw materials was measured as follows: the lignin content was determined according to the Klason method based on an initial 72% H 2 SO 4 hydrolysis step, followed by dilution with water to 3% H 2 SO 4 and reflux boiling in the secondary hydrolysis step ( Theander and Westerlund, 1986 Theander and Westerlund, 1986 ). The acid-insoluble residue (AIR) was calculated by the difference between the mass of the acid detergent-insoluble residue and the residual mass after the treatment. The presence of acid-soluble lignin (ASL) in the first filtrate was determined by ultraviolet-visible (UV-Vis) spectroscopy. Total cellulose content of the invasive species was investigated with the Kürschner-Hoffer method, which is based on the treatment of fibers with a mixture of nitric acid and ethanol (1:4, volume ratio) for a total duration of 4 h ( Béakou et al., 2008 Béakou et al., 2008 ). The hemicellulose content ( C h , %) was estimated theoretically as: C h =100- C c - C l , where C c is cellulose content (%) and C l is lignin content (%). An average of three replicates was calculated for each sample. 2.3.2　Morphology analysis 3 The morphology of the obtained freeze-dried nanocellulose samples was characterized with a scanning electron microscope at an accelerating voltage of 15 kV (Analytical JEOL 7001F FEG-SEM, JEOL, Tokyo, Japan). All samples were sputter-coated with a thin layer of Au before observation. Additional scanning electron microscopy (SEM) images and the energy-dispersive X-ray spectroscopy (EDX) analysis of the raw materials were obtained on a desktop SEM Phenom-ProX (ThermoFisher Scientific, London, UK). Samples were observed in their natural state, i.e., without any coating, using a Phenom Charge Reduction Sample Holder (ThermoFisher Scientific). The hydrodynamic diameter distribution and zeta potential of the obtained cellulose nanofibers were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively, using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) at 25 °C before the freeze-drying process. Three measurements were taken for each case; the mean value and standard deviation are reported. 2.3.3　X-ray diffraction and crystallinity studies 3 X-ray diffraction (XRD) studies of the raw materials and the freeze-dried cellulose nanofibers were carried out using a Bruker X8 advanced diffractometer (Bruker, Leipzig, Germany). Samples were scanned in the range of 5°–52° for 2 θ with increments of 0.02° using CuKα radiation ( λ =1.54 Å=0.154 nm). The CI of the samples was calculated by the Segal method ( Gümüskaya et al., 2003 Gümüskaya et al., 2003 ): CI=( I 200 - I am )/ I 200 ×100%, where I 200 gives the maximum intensity of the peak corresponding to the plane in the sample with Miller index of 200, and I am represents the intensity of diffraction of the non-crystalline material taken at an angle of 2 θ =18° in the valley between the peaks. The crystallite size ( L ) perpendicular to the plane was obtained by the Scherrer equation ( Poletto et al., 2014 Poletto et al., 2014 ): L =0.94 λ / β cos θ , where λ is the X-ray wavelength (0.1542 nm), β is the full width at half-maximum in radians, and θ is the Bragg angle in radians. 2.3.4　Fourier transform infrared spectroscopy 3 The Fourier transform infrared (FTIR) spectra of the freeze-dried cellulose nanofibers were analyzed using a Spectrum Two TM spectrophotometer (Perkin-Elmer, Massachusetts, USA). The spectra were obtained from KBr pellets containing the samples (measured between a wavenumber range of 4000 to 400 cm -1 ) at a scan rate of 32 scans/min in the transmittance mode. 2.3.5　Cell viability assay 3 The cytotoxic effects of the CNF AA1 , CNF AA2 , CNF RC1 , and CNF RC2 samples on human embryonic kidney 293T (HEK293T) cells were evaluated by thiazolyl blue tetrazolium bromide (MTT) colorimetric assay, establishing a correlation between cell metabolic activity and the number of viable cells in culture. Cell viability, which was studied as a function of the nanofiber type and its concentration, was assessed after exposing cells to various samples for 48 h. MTT was purchased from Sigma-Aldrich (Lisbon, Portugal), and where applicable, was used without further purification. Collagen (collagen I rat tail protein), Dulbecco's modified Eagle's medium (DMEM)‍-high glucose, fetal bovine serum (FBS), and antibiotic-antimycotic (AbAm) were purchased from Gibco ® (ThermoFisher Scientific), while analytical-grade dimethyl sulfoxide (DMSO) was purchased from ThermoFisher Scientific. Prior to cell cultivation, a 96-well plate was pre-treated with collagen (type I, 0.2 mg/mL in 0.25% (volume fraction) acetic acid). Thereafter, HEK293T cells (ATCC ® CRL-3216 TM ) were seeded in a 96-well plate at a density of 5×10 3 cells/well using 200 μL DMEM supplemented with 10% FBS and 1% AbAm solution. The cells were cultured for 24 h at 37 °‍C in a humidified 5% CO 2 atmosphere. At this stage, the cell culture medium in each well was replaced with 180 μL of fresh serum-containing DMEM, and the cells were subsequently exposed to 20 ‍μL of each cellulose nanofiber. Here, a 5 ‍mg/mL stock solution of the nanofiber was first prepared by suspending it in a 5% DMSO solution and then diluting it using filter-sterilized ultrapure water to different concentrations. Final concentrations of 0, 50, 100, 200, 250, 300, 400, and 500 μg/mL were tested for each cellulose nanofiber under investigation. After 48 h of incubation at 37 °‍C in a humidified 5% CO 2 atmosphere, the cell culture medium in each well was removed and replaced with a solution made up of 180 μL serum-containing DMEM and 20 μL MTT (5 mg/mL). The cells were further incubated for 4 h at 37 °‍C in a humidified 5% CO 2 atmosphere. The resultant medium in each well was then removed, and 100 μL DMSO was added to each well. This was followed by gentle shaking of the plate to help dissolve the purple MTT formazan crystals. Finally, the absorbance of each well was measured using a microplate reader (model Victor3 1420, PerkinElmer, Massachusetts, USA); the reference wavelength was set at 630 nm, and the absorbance at 490 nm was recorded. Three separate cell viability assays were conducted, where in each case, three replicates were analyzed for each sample under investigation. 3 Results and discussion 1 3.1 Chemical composition of raw materials 2 Chemical composition analysis showed that raw dried A. americana is composed of (33.2±1.8)% cellulose, (9.0±0.8)% lignin, and (12.1±0.6)% hemicellulose (all at mass fraction). Raw dried R. communis is composed of (34.4±1.8)% cellulose, (18.1±2.2)% lignin, and (32.9±4.3)% hemicellulose. The obtained results are comparable to data reported for other common lignocellulosic materials ( Phanthong et al., 2018 Phanthong et al., 2018 ). Nevertheless, it is important to mention that the chemical composition of the plants significantly depends on many different factors like geographic location, climate, harvesting, and the age of the plant, as well as the extraction process and experimental conditions. 3.2 Morphology characterization of the obtained samples 2 The surface morphologies of the dried milled plants of A. americana and R. communis were investigated by SEM in conjunction with EDX analysis (Figs. 2 2 and 3 3 ). As can be seen from Fig. 2 Fig. 2 a, SEM images of raw A. american a (raw_AA) showed two different morphologies. One part of the samples had a layered structure, while the other part formed a network of microfibrils tightly packed together and covered with some outer membranes. Closer examination of the raw_AA microfibrils revealed the diameter of the original fibril to be around 3 μm, with the ribbon-shaped fibrils having a spiral appearance. EDX analysis studies also revealed a homogeneous elemental composition throughout the whole fiber region, with several minerals commonly found in soil being identified (e.‍g., potassium and calcium; Fig. S1). The sample was very beam-sensitive, and the fibers were ultimately melted down and destroyed by the EDX electron beam. Fig. 3 Fig. 3 a illustrates the morphology of the raw R. communis samples (raw_RC). It is evident that the structure consists of spiral-shaped microfibrils covered with some non-cellulosic constituents (hemicellulose and lignin) ( Sair et al., 2017 Sair et al., 2017 ). Nanocellulose isolation was performed using the two different processes described above. Figs. 2b 2b and 2c 2c and Figs. 3b 3b and 3c 3c show the SEM micrographs of the freeze-dried cellulose nanofibers extracted from A. americana and R. communis fibers, respectively. The results indicated that both extraction methods contributed to structural changes in the cellulose nanofibers compared with the raw fibers. All synthesized samples exhibited a uniform and smooth morphology. We found that samples CNF AA1 and CNF RC1 extracted by method #1 had needle-shaped structures (Figs. 2b 2b and 3b 3b ). On the other hand, the CNF AA2 and CNF RC2 samples extracted by method #2 (based on sequential acid hydrolysis) showed a network with a structured fibrous morphology (Figs. 2c 2c and 3c 3c ). One of the main advantages of method #2 lies in the simplicity of the process since the application of the Kürschner-Hoffer method entails a rapid pre-treatment step for sample purification to remove the non-cellulosic components. The size distribution of the prepared samples measured by DLS is shown in Fig. S2. The cellulose nanofibers of the CNF AA1 and CNF AA2 samples had an average diameter of 58 and 141 nm, respectively, while the diameters of CNF RC1 and CNF RC2 were about 68 and 120 nm, respectively. As expected, the obtained average hydrodynamic size for all samples incorporated water in the primary hydration layers. 3.3 XRD characterization 2 The XRD results of the raw fibers and the extracted cellulose nanofibers are presented in Figs. 4 4 and 5 5 and Table 2 Table 2 . 2 Plane 2 Cellulose polymorph 6 2 θ (°) Raw_AA CNF AA1 CNF AA2 Raw_RC CNF RC1 CNF RC2 (1-10) Cellulose II 12.2 12.0 (1-10) Cellulose I 15.0 (110) Cellulose I 16.4 16.0 16.0 16.4 (110) Cellulose I 20.0 (112) Cellulose II 20.2 (020) Cellulose II 22.0 (200) Cellulose I 22.6 22.8 22.0 22.6 22.6 (004) Celluloses I and II 34.5 34.5 34.5 34.7 34.0 2 Crystallinity index (%) 70.4 87.7 94.1 90.7 92.7 2 Crystallite size (nm) 2.5 4.6 3.9 3.4 4.6 It should be noted that the structure and CI of the obtained samples depended on both the raw source of cellulose and the chemical extraction method applied. The raw A. americana fibers (i.e., raw_AA) and the cellulose nanofibers isolated from them had a crystalline nature ( Fig. 4 Fig. 4 ). The raw_AA sample, in particular, displayed typical cellulose I structure with characteristic diffraction peaks at 2 θ =16.4°, 22.6°, and 34.5°, indexed as (110), (200), and (004) planes, respectively ( Fig. 4 Fig. 4 ). The application of method #1 for CNF AA1 extraction from Agave fibers led to a change in the crystal form of the native cellulose I ( Fig. 4 Fig. 4 ). The CNF AA1 sample was a mixture of cellulose types I and II, as can be observed from the appearance of two peaks at 2 θ =20.0° (110) and 22.0° (020), instead of the one main peak at 22.6°. The obtained results were in agreement with previous studies ( le Moigne and Navard, 2010 le Moigne and Navard, 2010 ; Sghaier et al., 2012 Sghaier et al., 2012 ). In particular, Sghaier et al. (2012) Sghaier et al. (2012) reported a transformation of the crystal structure of A. americana fibers from cellulose I to cellulose II caused by mercerisation with NaOH, while bleaching with NaOCl treatment had no crucial effect on the Agave fiber properties. On the other hand, the CNF AA2 sample isolated by method #2 had a similar diffraction pattern to that of the raw Agave fibers ( Fig. 4 Fig. 4 ). For this sample, the 2 θ peaks were observed at 16.0° (110), 22.8° (200), and 34.5° (004), corresponding to the cellulose structure type I. For the A. americana fibers, both extraction methods contributed to a significant decrease in the amount of amorphous content in the samples. Nevertheless, as shown by the data reported in Table 2 Table 2 , the chemical treatment of the raw Agave fibers by method #2 significantly increased the CI of the cellulose nanofibers compared to extraction method #1. The calculated CIs of the raw_AA, CNF AA1 , and CNF AA2 samples were found to be 70.4%, 87.7%, and 94.1%, respectively. Das et al. (2010) Das et al. (2010) obtained crystallinity values of 66.5% for cotton nanofibers and 78.0% for jute, with crystallite sizes of 2.58 and 3.68 nm, respectively (perpendicular to the 101 plane); nanocrystalline cellulose obtained by acid hydrolysis from newspapers reached a CI of up to 90.2%, with a crystallite size of 5.7 nm ( Mohamed et al., 2015 Mohamed et al., 2015 ). The average crystallite size was calculated using the Scherrer formula. Estimation of the sizes of raw_AA, CNF AA1 , and CNF AA2 resulted in values of 2.5, 4.6, and 3.9 nm, respectively. The X-ray diffraction patterns of the raw_RC, CNF RC1 , and CNF RC2 samples are presented in Fig. 5 Fig. 5 . As can be seen, the diffraction pattern of raw R. communis fibers (i.e., raw_RC) is dominated by a broad, amorphous halo, showing a broad peak at around 22.0° in the (200) plane, typical for cellulose type I ( Fig. 5 Fig. 5 ). The X-ray diffraction diagram of CNF RC1 showed three characteristic peaks at 16.0° (110), 22.6° (200), and 34.7° (004), typical of the cellulose I structure ( Fig. 5 Fig. 5 ). Compared to CNF AA2 , the pattern of the CNF RC2 sample extracted using method #2 exhibited peaks at 2 θ =12.0° (1‒10), 15.0° (1‒10), 16.4° (110), 20.2° (112), 22.6° (200), and 34.0° (004), indicating a mixture of the cellulose types I and II crystal structures ( Fig. 5 Fig. 5 ). Nanocrystalline cellulose from cotton shows characteristics peaks at around 15.0° (110) and 23.0° (200), similar to what is observed here. Peaks at 2 θ =14.8°, 16.4°, 22.6°, and 34.6° are typical of cellulose I, while the one at 22.6° indicates a higher perfection of the crystal structure in the (200) plane ( Mohamed et al., 2015 Mohamed et al., 2015 ); this peak is found for samples with higher crystallinity values. The CI value of CNF RC1 was found to be 90.7%. After extraction by method #2, the CI of CNF RC2 was slightly higher (i.e., 92.7%). Estimation of the sizes of CNF RC1 and CNF RC2 resulted in values of 3.4 and 4.6 nm, respectively. These results showed that for both A. americana and R. communis fibers, extraction method #2 was better at producing highly crystalline cellulose nanofibers once ethanol at high temperatures had dissolved lignin and nitric acid had destroyed hemicellulose. 3.4 FTIR spectroscopy 2 The FTIR spectra of the CNF AA1 , CNF AA2 , CNF RC1 , and CNF RC2 samples are presented in Fig. 6 Fig. 6 . The broad band positioned in the 3600‍–‍3000 cm -1 region was observed in all the synthesized cellulose fiber samples and indicates the O‍–‍H stretching vibrations of the hydrogen-bonded hydroxyl group in the cellulose molecules involved in intermolecular hydrogen bonds ( Poletto et al., 2014 Poletto et al., 2014 ). The absorption peak at 1642 сm -1 in all the synthesized samples indicates the presence of water, due to the presence of O‍–‍H bending. Although all the FTIR samples were freeze-dried before analysis, it was difficult to completely eliminate water from the cellulose molecules due to the strong cellulose–water interactions. One of the possible explanations is that the open surfaces created in the nanocellulose by the removal of lignin and hemicellulose helped in the absorption of moisture, leading to higher moisture content ( Lam et al., 2012 Lam et al., 2012 ). The presence of the absorption peak at 2900 cm -1 can be attributed to the stretching vibrations of the C–H groups of cellulose ( Bettaieb et al., 2015a Bettaieb et al., 2015a ). The absorption peak in the spectrum near 1159 cm -1 is attributable to the C–O–C asymmetric stretching vibrations of cellulose. Several characteristic absorption bands showed that there were differences in the chemical structure of the cellulose nanofiber samples obtained by the two different extraction methods. This was the case for CNF AA1 and CNF RC1 samples isolated by method #1. The weak shoulders at 2854 cm -1 or/and 1740 cm -1 in the FTIR spectra are indicative of the presence of lignin content. In the case of the CNF AA2 and CNF RC2 samples isolated by method #2, these peaks were not detected. The band around 897 cm -1 is associated with an amorphous region in cellulose ( Hospodarova et al., 2018 Hospodarova et al., 2018 ) (β-glycosidic linkages ( Mohamed et al., 2015) Mohamed et al., 2015) ), which seems to be less important for R. communis samples and for Agave treated under method #2, which is in accordance with the results from crystallinity measures shown in Table 2 Table 2 . For all the synthesized samples, the presence of the symmetrical C‍–‍O‍–‍S vibration at 807 cm -1 is associated with the C‍–‍O‍–‍SO 3 groups. This result was confirmed by zeta potential measurements, where the negative ζ -potential values obtained for the CNF AA1 , CNF AA2 , CNF RC1 , and CNF RC2 samples can be explained by the presence of the negatively charged sulfate groups on the nanocellulose surface (Table S1). 3.5 Cytotoxicity assays 2 Cytotoxicity is an important issue that needs to be addressed before using any chemical compound or material on humans. Since the A. americana and R. communis cellulose nanofibers prepared in this study may be considered for future use in biomedical applications, their cytocompatibility with human cells was evaluated. As can be seen in Fig. 7 Fig. 7 , the cellulose nanofibers obtained using both extraction methods were all cytocompatible, as the HEK293T cells showed good viability. In the case of the cellulose nanofibers obtained using extraction method #1 (i.e., CNF AA1 and CNF RC1 ), the exposed cells were capable of actively metabolizing MTT in the concentration range assayed (i.e., 0–500 μg/mL). For the nanofibers obtained using extraction method #2 (i.e., CNF AA2 and CNF RC2 ), the cells were also observed to actively metabolize MTT in the same concentration range, but to a lesser extent relative to their method #1 counterparts (i.‍e., CNF AA1 and CNF RC1 ); this may be related to the crystallinity (it seems that the higher the CI, the lower the MTT metabolization). Moreover, relative to the CNF AA1 , CNF RC1 , and CNF RC2 samples, only the CNF AA2 sample was observed to exert a mild cytotoxic effect on the HEK293T cells when using a sample concentration of 500 μg/mL ( < 80% cell metabolic activity). Interestingly, cell viability was shown to be higher than the control values in most situations, which likely reflects a release of glucose from the nanofibers over time and its use in cell metabolism. Overall, A. americana and R. communis cellulose nanofibers prepared in this study were shown to be cytocompatible, with the potential to be used for future biomedical applications. 4 Conclusions 1 The fibers of invasive species A. americana and R. communis can be successfully used as a new source to produce very highly crystalline cellulose nanofibers with a CI of 94.1% and 92.7%, respectively, by two different processes, based on a first step of alkaline or acid treatment. 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Int J Pharm , 370 ( 1-2 ): 160 - 166 . https://doi.org/10.1016/j.ijpharm.2008.12.008 https://doi.org/10.1016/j.ijpharm.2008.12.008 Novel drug delivery devices for providing linear release profiles fabricated by 3DP Experiments, data analysis, and original draft preparation: Olga L. EVDOKIMOVA; experiments, data analysis, and writing the manuscript: Carla S. ALVES and Radenka M. KRSMANOVIĆ WHIFFEN; experiments, data analysis, funding acquisition, resources, and writing and editing the manuscript: Zaida ORTEGA; data analysis, and writing and editing the manuscript: Helena TOMÁS; conceptualization, supervision, funding acquisition, resources, data analysis, and writing and editing the manuscript: João RODRIGUES. All authors have read and agreed to the published version of the manuscript. The authors have full access to all the data in the study and take responsibility for the integrity and security of the data. The authors acknowledge the Programa de Cooperación Territorial INTERREG V-A MAC 2014-2020 and Inv2Mac Project (MAC/4.6d/229), as well as the partial support of FCT-Fundação para a Ciência e a Tecnologia (Base Fund UIDB/00674/2020). ARDITI-Agência Regional para o Desenvolvimento da Investigação Tecnologia e Inovação supported the study through the project M1420-01-0145-FEDER-000005-CQM+ (Madeira 14-20 Program); the Post-doc Grant (M1420-09-5369-FSE-000001, 002458/2015/132) for Carla S. ALVES is also acknowledged. 10.1631/jzus.B2000683 1673-1581（2021）06-0450-12 2020-10-24 2021-01-08 2021-06-15 2022-05-09 2021-04-12 © Zhejiang University Press 2021 Olga L. EVDOKIMOVA, Carla S. ALVES, Radenka M. KRSMANOVIĆ WHIFFEN, Zaida ORTEGA, Helena TOMÁS, and João RODRIGUES declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 6 22