Article Journal of Biomedical Nanotechnology
Copyright © 2013 American Scientific Publishers All rights reserved Printed in the United States of America
Vol. 9, 1293–1298, 2013 www.aspbs.com/jbn
Shape Control of Cellulose Nanocrystals via Compositional Acid Hydrolysis Changyoon Baek1 , Zahid Hanif2 , Seung-Woo Cho3 , Dong-Ik Kim4 , and Soong Ho Um1 5 ∗ 1 Department of Chemical Engineering and 5 SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do, 440-746, Korea 2 Department of Materials Science and Engineering, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju, 500-712, Korea 3 Department of Biotechnology, Yonsei University, 50, Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea 4 Division of Vascular Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Irwon-dong, Gangnam-gu, Seoul, 135-710, Korea
The current medical technology has constantly demanded novel and advanced materials exhibiting the unique physicochemical properties but, at the same time, possessing the intrinsic biocompatibility. Natural biomolecules based on materials such as peptide- (or protein-) or DNA/RNA derivatives have been formerly considered. To date, a carbohydrate-derived Delivered by Publishing Technology to: which Rice University molecule has been a highlight as a substitute with the prior biomaterials, suffer from their sequence-dependent IP: 210.38.137.106 On: Thu, 25 Feb 2016 16:50:35 immuno-cytoxicity. Of most, cellulose based materials have had a profound interest due to the great mechanical and Copyright: American Scientific Publishers optochemical properties as well as its immune-friendliness. However, it has been further manipulated in order to get distinctive structures at the desired shape and scale. Here, we report the versatile and synthetic technique to prepare spherical or rod-like cellulose nanocrystals (CNC). Under the varying concentrations of strong sulfuric acids (H2 SO4 and hydrochloric acids (HCl), spherical or rod-typed CNCs were selectively manufactured via acid hydrolysis of microcrystalline celluloses (MCC) in massive bundles. At the sequential addition of H2 SO4 and HCl in 1 to 2.5 molar ratios, most interestingly, the spherical CNC alone was observed with the average size of 50 nm in narrow distribution. All of CNCs had the larger surface area with mesoporosity. In addition, it was confirmed by the crystallographic measurement that it was very similar to the maternal structure originating from the bundle celluloses. It is much anticipated that the porous cellulose nanocrystals may play a principal role as a potential drug carrier for diseased biological compartments.
KEYWORDS: Microcrystalline Cellulose (MCC), Cellulose Nanocrystals (CNC), Acid Hydrolysis, Spherical or Rod-Type Nanoparticulate, Mesoporous Structure, Drug Carrier.
INTRODUCTION Nanotechnology has been practically utilized for various medical research areas owing to its unique physiochemical characteristics including, for instance, size and shapedependent specific photo-sensitivity and optoselectivity.1 2 In addition, once incorporated with biocompatible polymeric groups, it has been capable of being one of the promising alternatives of in vivo drug delivery carriers.1 3 In general, some nanomaterials for medical applications ∗
Author to whom correspondence should be addressed. Email: [email protected] Received: 15 September 2011 Accepted: 28 March 2012
J. Biomed. Nanotechnol. 2013, Vol. 9, No. 7
may have some restricted requirements such as tolerable biosafety and intrinsic biocompatibility and, at the same time, they may be seriously considered with respect to the target specific and efficient drug loading.4 5 Based on these, inorganic nano-particulates may be disqualified due to the cytotoxicity under physiological condition.6 As an alternative, nature-derived materials such as nucleic acids, proteins, and car-bohydrates have been presented. Of most, the carbohydrate-based material is currently highlighted. In particular, cellulose is a leading resource owing to the intrinsic biocompatibility. The cellulose is a polysaccharide in which it contains repeated units formulated by (C6 H10 O5 n and it sequentially polymerizes via a ß (1 → 4) glycosidic bond. It features a flat ribbon-like
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spherical and highly porous CNC with 50 nm in size may conformation because the internal glucose units are tightly crosslinked laterally.7 Cellulose is stiff with a high tensile be simply applied to the in vivo drug delivery system. strength, which may be due to the hierarchical structures based on microfibrils containing inter-hydrogen bonds EXPERIMENTAL DETAILS between the hydroxyl groups of cellulose chains.8 The Materials microfibrils ranging from 3 to 20 nm in diameter and The commercial microcrystalline cellulose powder was several m in length, become a tree, a cotton and even purchased by SERVA (Heidelberg, Germany). The cellua bacteria. The microfibrils are composed of crystalline lose average size was reported around 0.02 mm according regions possessing highly arranged cellulose chains and to the manufacture protocol. For acid hydrolysis, sulfuconcurrently, amorphous regions containing randomly disric acid (98% w/w), hydrochloric acid (37% w/w) were 7 ordered cellulose chains. The microcrystalline cellulose used directly as provided by Daejung Chemicals Co., Ltd. (MCC) could be obtained by the removal of amorphous (Seoul, Korea), without more purifications. regions of high-qualified wood(-cellulose) pellets through the process of acid hydrolysis. MCC has several feaPreparation of Cellulose Nanocrystals (CNCs) tures including no toxicity, good hygroscopicity, chemical According to the environmental conditions provided here inactivity, and low cost. Therefore, it is widely used for (e.g., mixing concentrations of strong acids, temperamulti-purpose as a basic composite as well as a billiontures), it was simply categorized into CNC series like, for dollar pharmaceutics.9 MCC may be further manipulated instance, CNC 1. For example, at the case of CNC 1 to 4, by strong acid hydrolysis in order to obtain the smaller microcrystalline celluloses (MCCs, 10 g) were dropped nanocrystals based on only celluloses (CNC). Its crysinto 200 ml of each acid or mixtures (at the volume ratio tallinity is usually very high up to 65–95%.7 10 The comH2 SO4 : HCl is of 3:1) under other temperatures. The more positional strong acids induce the break-up of glycosidic protocol was as reported in the early literature.11 At the bonds in amorphous regions and the neighboring water case of CNC 5, MCCs (10 g) were mixed together with molecules easily penetrate into the unsecured amorphous 200 mL of hydrochloric acid alone. It was successively and regions. According to several factors including acid confurther hydrolyzed by supplementary 200 mL of sulfuric centration, acid composition, reaction time and environacids. to: Rice University Publishing Technology mental temperature, the finalDelivered shapes of by CNC were quite 210.38.137.106 Feb 2016the 16:50:35 more detailed experimental conditions variable.11–14 With the intrinsic IP: biocompatibility and On: smallThu, 25Regarding Copyright: American Scientific Publishers of acid hydrolysis described above, it was shown at the size variation, CNC have been endeavored toward biomedTable I. Briefly, MCCs were gently stirred with acid mixical fields even though its physiochemical characteristics tures and they ended with the addition of 5-fold volare still under the investigation.15–19 It is clearly eliminated ume of deionized water. The reactant suspensions were by human body, as yet relying on its shape.20 21 Denrepeatedly (more than three times) washed with deionized nis and his colleagues demonstrated that filament-shaped water and then centrifuged down (HA-1000-3, HANIL, CNC nanoparticles could circulate in the blood by approxKorea) at 3,000 rpm for 5 minutes. The supernatant was imately ten-fold longer time relative to those of spherremoved and replaced by fresh deionized water. This washical ones.22 Moreover, the CNC nanoparticle uptake by ing step was repeated until the supernatant became turbid. macrophage occurred in the liver and spleen when its size 23 24 The aggregates remaining in suspension were disrupted by was over 100 nm. Therefore, the spherical CNC with Ultrasonification (Ultra sonic Processor, Sonic and Materithe diameter of less than 100 nm may be on demand for als Inc, USA) at 35 KHz for 5 minutes. The CNC suspenefficient medical applications. However, it has not been sion was subsequently frozen by liquid nitrogen (N2 in an achieved yet. In this paper, we report that CNC were synthesized in the spherical shape of 50 nm size. By using the compoTable I. The conditions of acid hydrolysis. CNC 3 was presitional mixtures of hydrochloric acids and sulfuric acids, treated 5 M NaOH for 4 h. it could be possible to control the shape. The final shape Hydrolysis condition of CNC was confirmed by using Transmission Electron Acid composition Microscopy (TEM) and Atomic Force Microscopy (AFM). Sample Step H2 SO4 HCl Temp. ( C) Time (h) The size was reassuringly measured by Dynamic Light Scattering (DLS). The inclusion capability of spherical CNC 1 1 48% – 50 3 CNC was inversely calculated through the pore size and CNC 2 1 98% 37% 50 3 the surface area as measured by using Brunauer–Emmett– CNC 3 1 98% 37% 50 3 CNC 4 1 – 37% 50 3 Teller (BET) method and Barrett–Joyner–Halenda (BJH) CNC 5 1st – 12.4% 80 4 method respectively. Irrespective of its shapes, spherical 2nd 55% – 40 2 and rod-like CNCs had slightly resembled each other on CNC 6 1 65% – 70 1 the pore volume similarity of 90 %. It is speculated that 1294
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Shape Control of Cellulose Nanocrystals via Compositional Acid Hydrolysis
ice-tray. The ice cube forms were put into a freeze-dryer (Bondiro, Ilshin Lab Co., Ltd., Korea) for 4 days. Characterization of Cellulose Nanocrystals (CNCs) To evaluate the size distribution, the sample was dissolved into 1 mL of deionized water and it finally reached the concentration of 0.02 wt%. The size of nanocrystlas was measured using a dynamic light scattering (DLS, ELS-8000, Otsuka Electronics Co., Japan) under the environmental temperature of 25 C and with the repetition time set to 10 runs. The crystal structure of CNCs was investigated by X-ray diffractometer (XRD, PANalytical, Netherlands). The shape and size of CNCs were determined by Transmission Electron Microscopy (TEM, JEM2100, JEOL Ltd., Japan) and Atomic Force Microscopy (non-contact mode AFM, XE-100, Park System, Korea). The surface areas were measured by Brunauer–Emmett– Teller (BET) method based on N2 adsorption-desorption. The pore size and volume were analyzed by Barrett– Joyner–Halenda (BJH) method.
RESULTS AND DISCUSSION
Figure 1. Size evaluation of synthetic cellulose nanocrystals: (a) CNC 1, (b) CNC 2, (c) CNC 3, (d) CNC 4, (e) CNC 5, (f) CNC 6. The bar charts show the size distribution of cellulose nanocrystals prepared under different acid hydrolytic conditions.
External Morphology and Internal Structure of Cellulose Nanocrystals (CNCs) by the result, we struggled with serial hydrolysis with both hydrochloride and University sulfuric acid. It finally resulted in CNC The morphology and structure Delivered of cellulose nanocrystals by Publishing Technology to: Rice particles of 2016 average-sized were consequently affected by IP: the 210.38.137.106 varying concentraOn: Thu, 25 Feb 16:50:3550 nm with narrow distribution (Fig. 1(e), Publishers in the case of CNC 5). Compared with the optitions and compositions of strong acid Copyright: mixtures. Table I American Scientific mized condition of sulfuric acid alone, it demonstrated that represented the different environmental conditions for the the CNC size in Figure 1(e) was 2-times less than that of formation of cellulose nanocrystals. Under the different Figure 1(f). conditions, CNCs were synthesized. Figure 1 showed the The crystalline structures of commercial MCC and synsize distribution of CNCs obtained by various acid comthesized CNCs were relatively compared via the X-ray positions. The average size of the commercial MCC which diffractometer. Irrespective of the variations of the used are also termed as bundle cellulose, is approximately acids and environmental temperatures, the X-ray pattern 20 m. After the hydrolytic treatment, the size of all CNCs was almost the same in any case. As can be seen, was reduced. It indicated that strong acids may be highly both MCC and CNCs represented the highest value at effective to the decomposition of the glycosidic bonds 22.6 , at which it usually identify the distinctive crysamong polymer units, resulting in the smaller cellulose tal structure of the cellulose. Even after the treatment of particles. strong acids, it was confirmed that the crystal structure of In comparison with the final size of CNCs treated by CNCs remained similar to that of maternal MCC. Most only a sulfuric acid (in the case of CNC 1) or a mixture importantly, it was evaluated that the characteristic peak at of the sulfuric acid and hydrochloric acid at the volume 22.6 of Figure 2(a) (defining the commercial MCC) and ratio of 3:1 (in the case of CNC 2 and 3), interestingly, it Figure 2(b) was sharper than that shown in Figure 2(c), was proven that the supplementary hydrochloric acid may which may be very comparable to that reported by Rosa be fully cooperative to formulate the smaller particles. In et al.26 It indicated that the crystallinity of cellulose addition, the NaOH at the early step of a hygroscopiccrystals synthesized was restored even after the later proinducing reaction had no effect on the size change as cessing of acid hydrolysis along with the removal of the shown in Figures 1(b) and (c). There was the smaller size amorphous regions remaining in MCC structures. of CNCs of which range was from 50 to 150 nm. It implied The external morphology of CNCs was observed by that the smaller size may be due to the hygroscopic celvarious electron microscopes including TEM and AFM. lulose expansion caused by water absorption. It may be Figure 3 exhibited the TEM images of CNC series: CNC 1 proven that the hydrochloric acid made stabilize the MCC (Fig. 3(a)), CNC 2 (Fig. 3(b)), CNC 3 (Fig. 3(c)) and state in solution.25 In view of Figure 1(d) (in the case of CNC 4 (Fig. 3(d)). It was verified that CNC 1–4 were CNC 4), once hydrolyzed by the hydrochloric acid alone, the reaction proceeded slowly relative to others. Inspired rod-type with the different sizes as depicted by length and J. Biomed. Nanotechnol. 9, 1293–1298, 2013
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Figure 2. Crystalline analysis of MCC and CNCs: (a) Commercial MCC, (b) CNC under strong acid hydrolysis, (c) CNC under weak acid hydrolysis.
width of one rod in the Figure; CNC 1 at 200–500 nm by 10–50 nm, CNC 2 at 100–400 nm by 10–70 nm, CNC 3 at 100–200 nm by 10–20 nm and CNC 4 at 500–1500 nm by 20–100 nm respectively. It matched well with the previous size measurements. The sizes were broadly distributed with the decreasing concentration of sulfuric acid. It proved that sulfuric acid supplemented with hydrochloric acid was considerably effective as compared with the use of hydrochloric acid alone. More detailed size information about CNCs was observed by AFM. Figure 4 revealed that the size of CNCs was slightly larger than TEM images, but overall shape of CNCs existed in rodlike ones. Most interestingly, approximately 50 nm size Delivered by Publishing Technology to: Rice University of the spherical CNC was observed in narrow distribuIP: 210.38.137.106 On: Thu, 25 Feb 2016 16:50:35 tion under conditions of both Figures 4(e) and (f). These Scientific Publishers Copyright: American images well supported the results of the DLS measurement. In Figure 4(g), CNCs existed in particle aggregates with about 20 m in size and about 32.42 nm in height. Porosity and Surface Area of Cellulose Nanocrystals (CNCs) The porosity and surface area of CNCs (including both rod and spherical shapes) were measured by the N2 adsorption-desorption method. The pore area was calculated according to the principle that the total volume of the adsorbed area was converted into the volume of liquid. In
Figure 3. TEM images of cellulose nanocrystals: (a) CNC 1, (b) CNC 2, (c) CNC 3, (d) CNC 4. The scale bar is 100 nm.
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Figure 4. External morphologic observation of cellulosenanocrystals under AFM: (a) CNC 1, (b) CNC 2, (c) CNC 3, (d) CNC 4, (e) a single particle of spherical CNC 5 with the scan size of 10 m, (f) a single particle of spherical CNC 5 with the scan size of 20 m, (g) aggregates of spherical CNC 5 with the scan size of 20 m as indicated by small arrow heads.
Table II, the surface areas of the rod-typed CNC and the spherical type CNC were measured at 2.2673 m2 /g and 2.0304 m2 /g respectively. It specified that the surface area of the spherical CNC was about 90% similar to that of the rod CNC. As well, Table II showed the pore volume and size of two types of the CNCs. Once investigated by the BJH method, the ratios of the average width and diameter of the rod-typed CNC and the spherical CNC were almost 1:2.6 and 1:2.5 respectively. It signified that the pore shape of both CNCs was similar. The pore volume of the spherical CNC came to nearly 84% approaching that of the rod-typed CNC. Also, the spherical CNC were approximately 93% of the pore width and 91% of the pore diameter which were very similar to the rod-typed CNC. It was, then, established that both all have similar pore volumes and areas. J. Biomed. Nanotechnol. 9, 1293–1298, 2013
Baek et al. Table II.
Shape Control of Cellulose Nanocrystals via Compositional Acid Hydrolysis
The surface area, pore surface area, BJH pore volume and pore size of cellulose nanocrystals.
Shape of CNC
BET surface area (m2 /g)
BJH adsorption surface area of pores (m2 /g)
BJH adsorption volume of pores (cm3 /g)
BJH adsorption average pore width (Å)
BJH adsorption average pore diameter (Å)
2.2673 2.0304
0.770 0.706
0.005138 0.004294
104.0599 97.0796
267.042 243.130
Rod Spherical
CONCLUSION
2. V. P. Chauhan, Z. Popovic, O. Chen, J. Cui, D. Fukumura, M. G.
Bawendi, and R. K. Jain, Fluorescent nanorods and nanospheres In this research, it was reported that the shape and size for real-time in vivo probing of nanoparticle shape-dependent tumor of cellulose nanocrystals can be tunable by the varying penetration. Angew. Chem. Int. Ed. 123, 1 (2011). compositions of strong acid mixtures and by the acid 3. T. M. Allen and P. R. Cullis, Drug delivery systems: Entering the hydrolysis of microcrystalline cellulose. Interestingly, the mainstream. Science 303, 1818 (2004). 4. A. El-Ansary and S. Al-Daihan, On the toxicity of therapeusize of CNCs, under the mixed or single acid hydrolytic tically used nanoparticles: An overview. J. Toxicol. 2009, 1 condition, was reduced. At the lower concentrations of (2009). strong acids, MCC could dissolve enough slowly into glu5. D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit, and cose moieties,11 whereas the higher concentrations disinR. Langer, Nanocarriers as an emerging platform for cancer therapy. tegrated the only glycosidic bonds among polymers. The Nature 2, 751 (2007). 6. B. Fadeel and A. E. Garcia-Bennett, Better safe than sorry: Underacid hydrolytic reaction may make it possible to gain standing the toxicological properties of inorganic nanoparticles manthe smaller particles successfully. With single strong acid ufactured for biomedical applications. Adv. Drug Deliv. Rev. 62, 362 alone, there was no substantial change in the particu(2010). late size. In the middle of the entire processing, in case 7. S. J. Eichhorn, Cellulose nanowhiskers: Promising materials for of CNC 3, NaOH helped increase hygroscopicity and advanced applications. Soft Matter. 7, 303 (2011). 8. Y. Habibi, L. A. Lucia, and O. J. Rojas, Cellulose nanocrystals: made water molecules uptaken inside the networked celChemistry, self-assembly, and applications. Chem. Rev. 110, 3479 lulose structures, and even the small size of nanoparticles (2010). were observed. It suggested that improving the solubil9. M. A. S. A. Samir, F. Alloin, and A. Dufresne, Review of recent Delivered Publishing to: Rice University ity of MCC may be a significant factor forbyobtaining the Technology research into cellulosic whiskers, their properties and their applica210.38.137.106 On: Thu, 25 Feb 2016 16:50:35 smaller sized and spherical typed IP: CNC. With the sequention in nanocomposite field. Biomacromolecules 6, 612 (2005). Copyright: American Scientific Publishers 10. P. Lu and Y. Hsieh, Preparation and properties of cellulose nanotial hydrolysis by hydrochloric acids and sulfuric acids, crystals: Rods, spheres, and network. Carbohydr. Polym. 82, 329 CNC produced regular spherical shapes in uniform size. (2010). In addition, it showed the large surface area similar to the 11. F. Camacho, P. Gonzalez-Tello, E. Jurado, and A. Robles, rod-typed CNC, also with higher mesoporosity. Microcrystalline-cellulose hydrolysis with concentrated sulphuric The spherical and porous CNC with 50 nm are strongly acid. J. Chem. Technol. Biotechnol. 67, 350 (1996). 12. X. Dong, J. O. Revol, and D. G. Gray, Effect of microcrystallite supposed to be a potential and promising drug delivery preparation conditions on the formation of colloid crystals of cellucargo owing to the membrane permeability and feasibility. lose. Cellulose 5, 19 (1998). Consequently, we are further testing its use as a biocom13. J. Zhang, T. J. Elder, Y. Pu, and A. J. Ragauskas, Facile synthepatible vehicle for several drugs and contrast reagents in sis of spherical cellulose nanoparticles. Carbohydr. Polym. 69, 607 the field of medicine. (2007).
Acknowledgment: The authors gratefully acknowledge support from the NCRC program of Korean Science and Engineering Foundation, which is being funded by the South Korean government (Grant No. R15-2008006-02002-0), and by the National Research Foundation of Korea (NRF) grant funded by the South Korean government (MEST) (Grant No. 20100007782; Midcareer Researcher Program), and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2010-0026793). REFERENCES 1. G. Orive, R. M. Hernández, A. R. Gascón, R. Calafiore, T. M. S. Chang, P. D. Vos, G. Hortelano, D. Hunkeler, I. Lacík, A. M. J. Shapiro, and J. Luis Pedraz, Cell encapsulation: Promise and progress. Nature Medicine 9, 104 (2003). J. Biomed. Nanotechnol. 9, 1293–1298, 2013
14. S. Elazzouzi-Hafraoui, Y. Nishiyama, J. Putaux, L. Heux, F. Dubreuil, and C. Rochas, The shape and size distribution of crystalline nanoparticles prepared by acid hydrolysis of native cellulose. Biomacromolecules 9, 57 (2008). 15. M. Hasani, E. D. Cranston, G. Westman, and D. G. Gray, Cationic surface functionalization of cellulose nanocrystals. Soft Matter. 4, 2238 (2008). 16. D. J. Gardner, G. S. Oporto, R. Mills, and M. A. S. A. Samir, Adhesion and surface issues in cellulose and nanocellulose. J. Adhes. Sci. Technol. 22, 545 (2008). 17. K. A. Mahmoud, J. A. Mena, K. B. Male, S. Hrapovic, A. Kamen, and J. H. T. Luong, Effect of surface charge on the cellular uptake and cytotoxicity of fluorescent labeled cellulose nanocrystals. ACS Appl. Mater. Interfaces 2, 2924 (2010). 18. S. D. and M. Roman, Fluorescently labeled cellulose nanocrystals for bioimaging applications. J. Am. Chem. Soc. 129, 13810 (2007). 19. M. Ka and M. B. H. S. L. Jh, Cellulose nanocrystal/gold nanoparticle composite as a matrix for enzyme immobilization. ACS Appl. Mater. Interfaces 7, 1383 (2009).
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24. S. M. Moghimi and J. Szebeni, Stealth liposomes and long circulating nanoparticles: Critical issues in pharmacokinetics, opsonization and protein-binding properties. Prog. Lipid Res. 42, 463 (2003). 25. J. Araki, M. Wada, S. Kuga, and T. Okano, Influence of surface charge on viscosity behavior of cellulose microcrystal suspension. J. Wood Sci. 45, 258 (1999). 26. M. F. Rosa, E. S. Medeiros, J. A. Malmonge, K. S. Gregorski, D. F. Wood, L. H. C. Mattoso, G. Glenn, W. J. Orts, and S. H. Imamb, Cellulose nanowhiskers from coconut husk fibers: Effect of preparation conditions on their thermal and morphological behavior. Carbohydr. Polym. 81, 83 (2010).
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Copyright © 2013 American Scientific Publishers All rights reserved Printed in the United States of America
Vol. 9, 1293–1298, 2013 www.aspbs.com/jbn
Shape Control of Cellulose Nanocrystals via Compositional Acid Hydrolysis Changyoon Baek1 , Zahid Hanif2 , Seung-Woo Cho3 , Dong-Ik Kim4 , and Soong Ho Um1 5 ∗ 1 Department of Chemical Engineering and 5 SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do, 440-746, Korea 2 Department of Materials Science and Engineering, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju, 500-712, Korea 3 Department of Biotechnology, Yonsei University, 50, Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea 4 Division of Vascular Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50, Irwon-dong, Gangnam-gu, Seoul, 135-710, Korea
The current medical technology has constantly demanded novel and advanced materials exhibiting the unique physicochemical properties but, at the same time, possessing the intrinsic biocompatibility. Natural biomolecules based on materials such as peptide- (or protein-) or DNA/RNA derivatives have been formerly considered. To date, a carbohydrate-derived Delivered by Publishing Technology to: which Rice University molecule has been a highlight as a substitute with the prior biomaterials, suffer from their sequence-dependent IP: 210.38.137.106 On: Thu, 25 Feb 2016 16:50:35 immuno-cytoxicity. Of most, cellulose based materials have had a profound interest due to the great mechanical and Copyright: American Scientific Publishers optochemical properties as well as its immune-friendliness. However, it has been further manipulated in order to get distinctive structures at the desired shape and scale. Here, we report the versatile and synthetic technique to prepare spherical or rod-like cellulose nanocrystals (CNC). Under the varying concentrations of strong sulfuric acids (H2 SO4 and hydrochloric acids (HCl), spherical or rod-typed CNCs were selectively manufactured via acid hydrolysis of microcrystalline celluloses (MCC) in massive bundles. At the sequential addition of H2 SO4 and HCl in 1 to 2.5 molar ratios, most interestingly, the spherical CNC alone was observed with the average size of 50 nm in narrow distribution. All of CNCs had the larger surface area with mesoporosity. In addition, it was confirmed by the crystallographic measurement that it was very similar to the maternal structure originating from the bundle celluloses. It is much anticipated that the porous cellulose nanocrystals may play a principal role as a potential drug carrier for diseased biological compartments.
KEYWORDS: Microcrystalline Cellulose (MCC), Cellulose Nanocrystals (CNC), Acid Hydrolysis, Spherical or Rod-Type Nanoparticulate, Mesoporous Structure, Drug Carrier.
INTRODUCTION Nanotechnology has been practically utilized for various medical research areas owing to its unique physiochemical characteristics including, for instance, size and shapedependent specific photo-sensitivity and optoselectivity.1 2 In addition, once incorporated with biocompatible polymeric groups, it has been capable of being one of the promising alternatives of in vivo drug delivery carriers.1 3 In general, some nanomaterials for medical applications ∗
Author to whom correspondence should be addressed. Email: [email protected] Received: 15 September 2011 Accepted: 28 March 2012
J. Biomed. Nanotechnol. 2013, Vol. 9, No. 7
may have some restricted requirements such as tolerable biosafety and intrinsic biocompatibility and, at the same time, they may be seriously considered with respect to the target specific and efficient drug loading.4 5 Based on these, inorganic nano-particulates may be disqualified due to the cytotoxicity under physiological condition.6 As an alternative, nature-derived materials such as nucleic acids, proteins, and car-bohydrates have been presented. Of most, the carbohydrate-based material is currently highlighted. In particular, cellulose is a leading resource owing to the intrinsic biocompatibility. The cellulose is a polysaccharide in which it contains repeated units formulated by (C6 H10 O5 n and it sequentially polymerizes via a ß (1 → 4) glycosidic bond. It features a flat ribbon-like
1550-7033/2013/9/1293/006
doi:10.1166/jbn.2013.1535
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Shape Control of Cellulose Nanocrystals via Compositional Acid Hydrolysis
Baek et al.
spherical and highly porous CNC with 50 nm in size may conformation because the internal glucose units are tightly crosslinked laterally.7 Cellulose is stiff with a high tensile be simply applied to the in vivo drug delivery system. strength, which may be due to the hierarchical structures based on microfibrils containing inter-hydrogen bonds EXPERIMENTAL DETAILS between the hydroxyl groups of cellulose chains.8 The Materials microfibrils ranging from 3 to 20 nm in diameter and The commercial microcrystalline cellulose powder was several m in length, become a tree, a cotton and even purchased by SERVA (Heidelberg, Germany). The cellua bacteria. The microfibrils are composed of crystalline lose average size was reported around 0.02 mm according regions possessing highly arranged cellulose chains and to the manufacture protocol. For acid hydrolysis, sulfuconcurrently, amorphous regions containing randomly disric acid (98% w/w), hydrochloric acid (37% w/w) were 7 ordered cellulose chains. The microcrystalline cellulose used directly as provided by Daejung Chemicals Co., Ltd. (MCC) could be obtained by the removal of amorphous (Seoul, Korea), without more purifications. regions of high-qualified wood(-cellulose) pellets through the process of acid hydrolysis. MCC has several feaPreparation of Cellulose Nanocrystals (CNCs) tures including no toxicity, good hygroscopicity, chemical According to the environmental conditions provided here inactivity, and low cost. Therefore, it is widely used for (e.g., mixing concentrations of strong acids, temperamulti-purpose as a basic composite as well as a billiontures), it was simply categorized into CNC series like, for dollar pharmaceutics.9 MCC may be further manipulated instance, CNC 1. For example, at the case of CNC 1 to 4, by strong acid hydrolysis in order to obtain the smaller microcrystalline celluloses (MCCs, 10 g) were dropped nanocrystals based on only celluloses (CNC). Its crysinto 200 ml of each acid or mixtures (at the volume ratio tallinity is usually very high up to 65–95%.7 10 The comH2 SO4 : HCl is of 3:1) under other temperatures. The more positional strong acids induce the break-up of glycosidic protocol was as reported in the early literature.11 At the bonds in amorphous regions and the neighboring water case of CNC 5, MCCs (10 g) were mixed together with molecules easily penetrate into the unsecured amorphous 200 mL of hydrochloric acid alone. It was successively and regions. According to several factors including acid confurther hydrolyzed by supplementary 200 mL of sulfuric centration, acid composition, reaction time and environacids. to: Rice University Publishing Technology mental temperature, the finalDelivered shapes of by CNC were quite 210.38.137.106 Feb 2016the 16:50:35 more detailed experimental conditions variable.11–14 With the intrinsic IP: biocompatibility and On: smallThu, 25Regarding Copyright: American Scientific Publishers of acid hydrolysis described above, it was shown at the size variation, CNC have been endeavored toward biomedTable I. Briefly, MCCs were gently stirred with acid mixical fields even though its physiochemical characteristics tures and they ended with the addition of 5-fold volare still under the investigation.15–19 It is clearly eliminated ume of deionized water. The reactant suspensions were by human body, as yet relying on its shape.20 21 Denrepeatedly (more than three times) washed with deionized nis and his colleagues demonstrated that filament-shaped water and then centrifuged down (HA-1000-3, HANIL, CNC nanoparticles could circulate in the blood by approxKorea) at 3,000 rpm for 5 minutes. The supernatant was imately ten-fold longer time relative to those of spherremoved and replaced by fresh deionized water. This washical ones.22 Moreover, the CNC nanoparticle uptake by ing step was repeated until the supernatant became turbid. macrophage occurred in the liver and spleen when its size 23 24 The aggregates remaining in suspension were disrupted by was over 100 nm. Therefore, the spherical CNC with Ultrasonification (Ultra sonic Processor, Sonic and Materithe diameter of less than 100 nm may be on demand for als Inc, USA) at 35 KHz for 5 minutes. The CNC suspenefficient medical applications. However, it has not been sion was subsequently frozen by liquid nitrogen (N2 in an achieved yet. In this paper, we report that CNC were synthesized in the spherical shape of 50 nm size. By using the compoTable I. The conditions of acid hydrolysis. CNC 3 was presitional mixtures of hydrochloric acids and sulfuric acids, treated 5 M NaOH for 4 h. it could be possible to control the shape. The final shape Hydrolysis condition of CNC was confirmed by using Transmission Electron Acid composition Microscopy (TEM) and Atomic Force Microscopy (AFM). Sample Step H2 SO4 HCl Temp. ( C) Time (h) The size was reassuringly measured by Dynamic Light Scattering (DLS). The inclusion capability of spherical CNC 1 1 48% – 50 3 CNC was inversely calculated through the pore size and CNC 2 1 98% 37% 50 3 the surface area as measured by using Brunauer–Emmett– CNC 3 1 98% 37% 50 3 CNC 4 1 – 37% 50 3 Teller (BET) method and Barrett–Joyner–Halenda (BJH) CNC 5 1st – 12.4% 80 4 method respectively. Irrespective of its shapes, spherical 2nd 55% – 40 2 and rod-like CNCs had slightly resembled each other on CNC 6 1 65% – 70 1 the pore volume similarity of 90 %. It is speculated that 1294
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ice-tray. The ice cube forms were put into a freeze-dryer (Bondiro, Ilshin Lab Co., Ltd., Korea) for 4 days. Characterization of Cellulose Nanocrystals (CNCs) To evaluate the size distribution, the sample was dissolved into 1 mL of deionized water and it finally reached the concentration of 0.02 wt%. The size of nanocrystlas was measured using a dynamic light scattering (DLS, ELS-8000, Otsuka Electronics Co., Japan) under the environmental temperature of 25 C and with the repetition time set to 10 runs. The crystal structure of CNCs was investigated by X-ray diffractometer (XRD, PANalytical, Netherlands). The shape and size of CNCs were determined by Transmission Electron Microscopy (TEM, JEM2100, JEOL Ltd., Japan) and Atomic Force Microscopy (non-contact mode AFM, XE-100, Park System, Korea). The surface areas were measured by Brunauer–Emmett– Teller (BET) method based on N2 adsorption-desorption. The pore size and volume were analyzed by Barrett– Joyner–Halenda (BJH) method.
RESULTS AND DISCUSSION
Figure 1. Size evaluation of synthetic cellulose nanocrystals: (a) CNC 1, (b) CNC 2, (c) CNC 3, (d) CNC 4, (e) CNC 5, (f) CNC 6. The bar charts show the size distribution of cellulose nanocrystals prepared under different acid hydrolytic conditions.
External Morphology and Internal Structure of Cellulose Nanocrystals (CNCs) by the result, we struggled with serial hydrolysis with both hydrochloride and University sulfuric acid. It finally resulted in CNC The morphology and structure Delivered of cellulose nanocrystals by Publishing Technology to: Rice particles of 2016 average-sized were consequently affected by IP: the 210.38.137.106 varying concentraOn: Thu, 25 Feb 16:50:3550 nm with narrow distribution (Fig. 1(e), Publishers in the case of CNC 5). Compared with the optitions and compositions of strong acid Copyright: mixtures. Table I American Scientific mized condition of sulfuric acid alone, it demonstrated that represented the different environmental conditions for the the CNC size in Figure 1(e) was 2-times less than that of formation of cellulose nanocrystals. Under the different Figure 1(f). conditions, CNCs were synthesized. Figure 1 showed the The crystalline structures of commercial MCC and synsize distribution of CNCs obtained by various acid comthesized CNCs were relatively compared via the X-ray positions. The average size of the commercial MCC which diffractometer. Irrespective of the variations of the used are also termed as bundle cellulose, is approximately acids and environmental temperatures, the X-ray pattern 20 m. After the hydrolytic treatment, the size of all CNCs was almost the same in any case. As can be seen, was reduced. It indicated that strong acids may be highly both MCC and CNCs represented the highest value at effective to the decomposition of the glycosidic bonds 22.6 , at which it usually identify the distinctive crysamong polymer units, resulting in the smaller cellulose tal structure of the cellulose. Even after the treatment of particles. strong acids, it was confirmed that the crystal structure of In comparison with the final size of CNCs treated by CNCs remained similar to that of maternal MCC. Most only a sulfuric acid (in the case of CNC 1) or a mixture importantly, it was evaluated that the characteristic peak at of the sulfuric acid and hydrochloric acid at the volume 22.6 of Figure 2(a) (defining the commercial MCC) and ratio of 3:1 (in the case of CNC 2 and 3), interestingly, it Figure 2(b) was sharper than that shown in Figure 2(c), was proven that the supplementary hydrochloric acid may which may be very comparable to that reported by Rosa be fully cooperative to formulate the smaller particles. In et al.26 It indicated that the crystallinity of cellulose addition, the NaOH at the early step of a hygroscopiccrystals synthesized was restored even after the later proinducing reaction had no effect on the size change as cessing of acid hydrolysis along with the removal of the shown in Figures 1(b) and (c). There was the smaller size amorphous regions remaining in MCC structures. of CNCs of which range was from 50 to 150 nm. It implied The external morphology of CNCs was observed by that the smaller size may be due to the hygroscopic celvarious electron microscopes including TEM and AFM. lulose expansion caused by water absorption. It may be Figure 3 exhibited the TEM images of CNC series: CNC 1 proven that the hydrochloric acid made stabilize the MCC (Fig. 3(a)), CNC 2 (Fig. 3(b)), CNC 3 (Fig. 3(c)) and state in solution.25 In view of Figure 1(d) (in the case of CNC 4 (Fig. 3(d)). It was verified that CNC 1–4 were CNC 4), once hydrolyzed by the hydrochloric acid alone, the reaction proceeded slowly relative to others. Inspired rod-type with the different sizes as depicted by length and J. Biomed. Nanotechnol. 9, 1293–1298, 2013
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Figure 2. Crystalline analysis of MCC and CNCs: (a) Commercial MCC, (b) CNC under strong acid hydrolysis, (c) CNC under weak acid hydrolysis.
width of one rod in the Figure; CNC 1 at 200–500 nm by 10–50 nm, CNC 2 at 100–400 nm by 10–70 nm, CNC 3 at 100–200 nm by 10–20 nm and CNC 4 at 500–1500 nm by 20–100 nm respectively. It matched well with the previous size measurements. The sizes were broadly distributed with the decreasing concentration of sulfuric acid. It proved that sulfuric acid supplemented with hydrochloric acid was considerably effective as compared with the use of hydrochloric acid alone. More detailed size information about CNCs was observed by AFM. Figure 4 revealed that the size of CNCs was slightly larger than TEM images, but overall shape of CNCs existed in rodlike ones. Most interestingly, approximately 50 nm size Delivered by Publishing Technology to: Rice University of the spherical CNC was observed in narrow distribuIP: 210.38.137.106 On: Thu, 25 Feb 2016 16:50:35 tion under conditions of both Figures 4(e) and (f). These Scientific Publishers Copyright: American images well supported the results of the DLS measurement. In Figure 4(g), CNCs existed in particle aggregates with about 20 m in size and about 32.42 nm in height. Porosity and Surface Area of Cellulose Nanocrystals (CNCs) The porosity and surface area of CNCs (including both rod and spherical shapes) were measured by the N2 adsorption-desorption method. The pore area was calculated according to the principle that the total volume of the adsorbed area was converted into the volume of liquid. In
Figure 3. TEM images of cellulose nanocrystals: (a) CNC 1, (b) CNC 2, (c) CNC 3, (d) CNC 4. The scale bar is 100 nm.
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Figure 4. External morphologic observation of cellulosenanocrystals under AFM: (a) CNC 1, (b) CNC 2, (c) CNC 3, (d) CNC 4, (e) a single particle of spherical CNC 5 with the scan size of 10 m, (f) a single particle of spherical CNC 5 with the scan size of 20 m, (g) aggregates of spherical CNC 5 with the scan size of 20 m as indicated by small arrow heads.
Table II, the surface areas of the rod-typed CNC and the spherical type CNC were measured at 2.2673 m2 /g and 2.0304 m2 /g respectively. It specified that the surface area of the spherical CNC was about 90% similar to that of the rod CNC. As well, Table II showed the pore volume and size of two types of the CNCs. Once investigated by the BJH method, the ratios of the average width and diameter of the rod-typed CNC and the spherical CNC were almost 1:2.6 and 1:2.5 respectively. It signified that the pore shape of both CNCs was similar. The pore volume of the spherical CNC came to nearly 84% approaching that of the rod-typed CNC. Also, the spherical CNC were approximately 93% of the pore width and 91% of the pore diameter which were very similar to the rod-typed CNC. It was, then, established that both all have similar pore volumes and areas. J. Biomed. Nanotechnol. 9, 1293–1298, 2013
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Shape Control of Cellulose Nanocrystals via Compositional Acid Hydrolysis
The surface area, pore surface area, BJH pore volume and pore size of cellulose nanocrystals.
Shape of CNC
BET surface area (m2 /g)
BJH adsorption surface area of pores (m2 /g)
BJH adsorption volume of pores (cm3 /g)
BJH adsorption average pore width (Å)
BJH adsorption average pore diameter (Å)
2.2673 2.0304
0.770 0.706
0.005138 0.004294
104.0599 97.0796
267.042 243.130
Rod Spherical
CONCLUSION
2. V. P. Chauhan, Z. Popovic, O. Chen, J. Cui, D. Fukumura, M. G.
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Acknowledgment: The authors gratefully acknowledge support from the NCRC program of Korean Science and Engineering Foundation, which is being funded by the South Korean government (Grant No. R15-2008006-02002-0), and by the National Research Foundation of Korea (NRF) grant funded by the South Korean government (MEST) (Grant No. 20100007782; Midcareer Researcher Program), and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2010-0026793). REFERENCES 1. G. Orive, R. M. Hernández, A. R. Gascón, R. Calafiore, T. M. S. Chang, P. D. Vos, G. Hortelano, D. Hunkeler, I. Lacík, A. M. J. Shapiro, and J. Luis Pedraz, Cell encapsulation: Promise and progress. Nature Medicine 9, 104 (2003). J. Biomed. Nanotechnol. 9, 1293–1298, 2013
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