Nanofibers of Cellulose and Its Derivatives Fabricated Using Direct Electrospinning

A short review with 49 references describes the electrospinninng (ES) process for polysaccharides, cellulose and chitosan, and their derivatives, including cellulose acetate and hydroxypropyl cellulose. A majority of applied studies adopted a two step-process, in which the cellulose acetate was used for the first ES process, followed by acetyl group removal to regenerate cellulose thin fibers. The electrospun nonwoven fabrics (ESNW) of regenerated cellulose can be modified by introduction of aldehyde groups by oxidative cleavage of vicinal diols using periodates, and these aldehyde groups serve as acceptors of foreign substances, with various chemical/biological functions, to be immobilized on the fiber surfaces in the ESNW matrices. Direct electrospinning of cellulose from trifluroacetic acid solution was also developed and the applied studies were summarized to conclude the current trends of interests in the ES and related technologies.

sheet-like materials fabricated via the ES process, with micro-scale structures involving a non-woven network of very thin fibers, are called "Electrospun Non-Woven Fabrics (ESNWs)". The original papers and review articles with various research purposes/motivations and studies have employed different terms for the electrospun fabrics, for instance, electrospun-(or nanofiber-) mats, -sheets, -non-woven films, -porous membranes, etc., thus in this article, the author will use the term 'ESNWs' to avoid any inconsistencies in the definition of the electrospun fabrics.
The crystalline polymers, including the naturally-occurring polysaccharides, cellulose and chitin, and chitosan are also much less soluble, and their available solvents are highly dielectric and not very volatile in many cases, and thus the known solvents of cellulose and chitosan are not routinely used for the ES process. These facts established an important objective in fine fiber technologies to determine the favorable solvents for the ES process of cellulose and chitosan. This article reviews the topics concerning the ES process of cellulose, chitin and their derivatives found in recent research publications. Table 1 summarizes the list of publications, mainly from 2010 to 2012, and the corresponding content previews of their topics, including material polymers, cellulose and derivatives, ES solvents, average diameters, and purpose of the research studies. In a strict sense, the terms 'nanofibers' and 'nano-scaled fibers' define those having average diameters less than 100 nm [3], however, most publications in Table 1 have applied the term 'nanofibers' for their fabrics, even in the cases where the average diameters were 200-800 nm (submicro-or micro-scaled). In addition to this inconsistency, several research studies in Table 1 include the phrase 'electrospun cellulose nanofiber' in their title when, in fact, the starting material used was CA, so that the readers should pay attention so as to not confuse the starting materials, the processing solvents, and the presence or absence of any pre-or post-processing of the ESNWs.
Since the first patents by Formhals in 1937 [1,33], the chemical conversion of CA-ESNW to RC-ESNW was only documented by Hsieh in 2002 [34], which means that it took almost 35 years to realize the successful fabrication of RC-ESNWs. There are now multiple ways to functionalize RC-ESNWs by a chemical reaction to generate aldehyde groups on the surface of the fine RC-ESNW fibers via oxidative cleavage of vicinal diols using an aqueous periodate solution, and the aldehyde groups work as acceptors of foreign ligands, including biologically active substances, enzymes, or affinity-targeted molecules, which are discussed in Section 3.1. Cellulose nitrates can be also fabricated as ESNWs [35].

'Direct' ES Process of Cellulose Solutions
In 2004, Ohkawa et al. published a research study on the ES of chitosan solution [36], the solute polymers of which have viscosity average molecular weights ranging from ca. 200,000 to 1,800,000, and then in 2006, the fabrication of chitosan-ESNW, of which the average diameters of single fine fibers were less than 100 nm, was realized [37], meaning that the first time "nanofiber chitosan-ESNW" came to the materials science of ES processing. Various combinations of organic acids were then examined as ES solvents, and among them, trifluoroacetic acid (TFA) was found to be the most favorable one for the ES-processing of chitosan [38]. Later in 2009, the authors found that cellulose-ESNW in which the average diameters of single fibers ranged from 100 nm to submicrometers, was fabricated when the TFA solvent was applied to the ES process of cellulose preparations from pulp and cotton [39]. This result has newly allowed the direct ES processing of cellulose preparations, without involving a cellulose derivative such as CA, and the authors called this one-step process without any pre-and post-spinning treatment 'direct' ES (dES).
Other research studies classified as dES employ aqueous N-methylmorpholine-N-oxide (NMMO aq.), which is a well-known solvent for regenerated cellulose fiber in industrial scale production, and a spruce cellulose preparation (Dp, 700-800) was fabricated as cellulose-ESNW (average diameter, 200-400 nm) in the presence or absence of the mercelization [40]. N-Alkylinidazolium-derivative ionic liquids (IL) have also been examined as processing solvents for the dES of cellulose [41,42] or a composite of cellulose and chitosan [43]. In the cases of dES processes using NMMO aq. and IL, the evaporation of solvent molecules might not occur, hence the fiber collector was a metallic plate immersed in water or alcohol. N,N-Dimethylacetamide containing ca. 8.0 wt % LiCl was also reported as a dES solvent in cellulose preparations, and after evaporation of the solvent, the remaining LiCl in the fiber matrix on the collector was removed by rinsing with water [11].
Other solvents for the ES process of cellulose can be found in several patent documents, but most of them describe solvents which are frequently used in the production of regenerated cellulose fiber. In several cases, the processes require pre-or post-ES spinning treatments. The dES of cellulose using perfluoro-organic acids [44] typically yields very fine fibers with the smallest diameter of 60-75 nm without any pre-or post-spinning treatment (Figure 1).

Chemical Modification of Cellulose-ESNWs
A review [4] describes in detail the applied studies of the fine fibers of organic polymers fabricated by ES processes. When compared to other fabrication methods, e.g., melt-browns, the lower production performance of the ES process is considered a problem, however, during the last decade, the problem has been solved with the development of suitable processing machinery for the large-scale fabrication of ESNWs, and hence, the scope and criteria of the applied studies of ESNWs has become wider and more varied.
One of the more remarkable functionalization strategies is chemical modification of the RC-ESNWs. Among the research studies listed in Table 1, periodates were used for the treatment of RC-ESNWs for generating aldehyde groups via oxidative cleavage of vicinal diols in the deacetylated glucopyranoside units. The aldehyde groups can immobilize various kinds of ligands, and thus the applied studies of RC-ESNWs encompass the scopes of: (i) scaffolds for cell culture; (ii) absorbents of heavy metal ions for healthcare or environmental technology; (iii) materials for the separation of conjugated and affinity linked bilogical substances; (iv) immobilized catalysts including enzymes; and (v) drug-releasing materials having antibacterial activities. A common feature of these objectives is utilization of the large surface area characteristic of the fine fiber ESNWs, and in some cases, a nano-scaled dispersion of the composite particles also having nanoscale sizes, while the biological and environmental compatibilities of cellulose are taken for granted.
Ohkawa et al., described the conjugation of ligands via an electrostatic interaction between hydroxypropyl cellulose (HPC)-ESNWs. The surfaces of the fine fibers in the HPC-ESNW matrices are chemically modified using diasocyanate cross-linkers ( Figure 2) [45]. The excess amount of the cross-linkers in the treatments plays two roles; one is just for cross-linking, which renders the HPC-ESNWs insoluble in most solvents, and the other one is for leaving unreacted NCO groups, which can be utilized both as: (a) a direct acceptor of any substance molecules having nucleophilic moieties, and (b) if N,N-diethylethylene diamine is used for the linkage via (a), strongly cationic (diethylaminoethyl-DEAE) groups can be introduced on the surfaces of the HPC fine fibers. These ideas have been derived from the method to prepare the DEAE-cellulose for immobilizing enzymes based on the original studied by Chibata et al. [46]. Aminocylase-I from porcine pancreas catalyzes the hydrolytic reaction of N α -acetyl-L-amino acids to yield the free L-amino acid, and the enzyme is highly active for L-Met and L-Leu. Figure 3. Chirality-specific hydrolysis of Ac-L-Met by aminoacylase-I-immobilized HPC-ESNWs. More details for experiments are described in reference [45]. Left, most all of Ac-L-Met fed were hydrolyzed; Middle, No hydrolysis was observed for Ac-D-Met except for decreases in peak area due to absorption; Right, Only Ac-L-Met was hydrolyzed in a racemic mixture. Co 2+ is a cofactor for enhancing the enzymatic activity.
When aminoacylase-I was immobilized on the DEAE-HCP-ESNWs, the immobilized aminoacylase-I exhibited a deacetylation catalysis with an L-specific chiral recognition towards L-Met, as seen for the free enzyme, and in repetitive cycles of use, the initial activity and L-specificity were mostly retained (Figure 3).

Cellulose-Composite-ESNWs
Another direction for applied research studies of the cellulose-ESNWs is to prepare cellulose-composite-ESNWs, and in these cases, cellulose derivatives are the major components, which carry subcomponents for functionalization, for instance, synthetic and natural polymeric materials, low molecular weight organic or inorganic substances, and carbon nanotubes for their applied studies from the viewpoints of electronic devices, hydrophobic (superabsorbent) polymers for healthcare and medical uses, antibacterial surface processing, reinforced composites, and sustained release materials. Most of the subcomponents were in the forms of nano-or submicro-scaled particles or whiskers and blended with the cellulose derivatives in the pre-spun solutions. A common feature in these studies is utilization of the chemical, mechanical, or electric properties of the subcomponents with a large surface area and nano-or micro-scaled dispersions/orientations.
When the shapes of the subcomponents are small sized whiskers or carbon nanotubes, a concentric spinneret is often employed to fabricate core-shell composite fine fibers [19,20,29]. References about both cellulose derivatives and coaxial electrospinning enable: (i) zero-order drug release cellulose acetate nanofibers; (ii) the preparation of high-quality ketoprofen-loaded cellulose acetate nanofibers; and (iii) electrospun biphasic drug release using polyvinylpyrrolidone/ethyl cellulose core/sheath nanofibers. Another fabrication method for composite ESNWs of polymers and cellulose-derivatives involves multi-nozzle jet emission, in which different kinds of polymer and cellulose derivative solutions were emitted from each of the independent nozzles by an applied voltage [6]. Among the research studies listed in Table 1, almost no dES methods were found, especially for the cases that cellulose itself (not its derivatives) is used as a major component. The reason for that is limitation in the choice of the ES solvent, whereby most of the available solvents, for example TFA, are highly acidic and strongly dielectric, and these properties will affect the chemical or electrical properties of the subcomponents. On the other hand, in the case of the CA-ESNWs, available solvents for the ES process are neutral, so that the stability of the subcomponents is not chemically damaged. The majority of researchers may be concerned with the preparation of the cellulose-composite-ESNWs being difficult, especially for biological subcomponents, including enzymes.
Ohkawa et al. have continued their applied studies on cellulose-composite-ESNWs using sub-components which are relatively stable, even in neat TFA solutions. Figure 4 is an example study for this approach, and the cellulose-ESNW subcomponent is a synthetic polypeptide, copoly[Ser(PO3H2) x , Asp y ] (x:y = 100:0, 75:25, 50:50, 25:75), on which serine residues are fully phosphorylated. The weight ratio of copoly[Ser(PO3H2) x , Asp y ] towards cellulose was 1.0-1.2 wt % in a TFA solution, and then after the ES process, the products, cellulose-copoly[Ser(PO3H2) x , Asp y ]composite-ESNW, was observed using a scanning electron microscope (Figure 4) [47]. The chemical structure of copoly[Ser(PO3H2) x , Asp y ] was designed on the basis of osteogenic proteins, of which their role is to induce the hydroxyapatite deposition to make hard tissues, teeth and bone. Since the synthesis route of the copolypeptide involves the final deprotection step in TFA, the ES solvent of the cellulose-copoly[Ser(PO3H2) x , Asp y ]-composite-ESNW does not affect the chemical structure of the subcomponent. A series of in vitro experimental results indicated that the composite ESNWs were able to induce hydroxyapatite deposition, which is associated with the crystalline polymorph transition similar to those occurred in vivo, and potential application of the cellulose-copoly[Ser(PO3H2) x , Asp y ]-composite-ESNWs is, hence, applied as the scaffolding materials for hard tissue engineering. As described above, reasonable design of the subcomponents and their chemical structures, which are derived from biological components, enables biomedical studies by the dES process and fabrication of cellulose-based-composite ESNWs [48,49].

On a Specific Issue of Cellulose-ESNWs Prepared from TFA Solutions
There is a specific issue when considering the potential applications of cellulose-ESNWs prepared via the dES process using TFA solutions. The term 'cellulose nanofiber' frequently appears in research articles, reviews and book chapters/sections, which interests are usually the I-type crystallites of cellulose, often referred to as 'cellulose nano-crystals (CNCs)' or 'cellulose nano-whiskers (CNWs)'. As already described in references [44,49], the cellulose-ESNWs prepared via dES from perfluoro-organic acids do not exhibit any of significant diffraction characteristic of I-III-type crystallites, which suggests that the cellulose molecules in the ESNW matrices adopt an amorphous state.
The fact mentioned above implies that the feature of the high modulus, I-type crystallites, is absent in the cellulose-ESNWs fabricated via the dES process. The directions of material design policies between the high modulus composites using CNCs or CNWs and the cellulose-nanofiber-ESNW via the dES process have to be distinguished on the basis of their principles. The latter cannot encompass the aim to develop a high modulus composite design, while the amorphous structures of cellulose matrices in the ESNWs inspire another policy to express the inclusion or intercalation properties of the cellulose-ESNWs, for instance, sustainable drug-release or the entrapment of heavy metal ions. The difference between the utilizations of the CNCs or CNWs and the electrospun cellulose nanofibers is remarkable, although both policies are classified in the developmental approach of 'cellulose nanofibers'.
Recently, a characteristic trial was performed to combine both features described above, and a reference [20] in Table 1 describes a fabrication method for cellulose-CNC-composite-ESNWs in which a double layered nozzle was employed to install the shell layer of a cellulose solution in NMMO aq. and the core layer of a CNC dispersion in dimethyl sulfoxide (DMSO). This combined approach will require several precise regulations, including the suppression of the CNC aggregation and the sufficient retention of the CNC weight fraction for significant reinforcement of the cellulose-CNC-composite nanofibers regarding their mechanical properties, while these conflicting features will not easily coincide, thus ensuring the preferable processing parameters, for instance, viscosity of the pre-spun dopes. The fundamental studies on the improved design of nozzles and on the reproducibility of the reinforcement effect for the modulus of every single nanofiber still remain to be investigated, as well as the combined approach, cellulose-CNC-composite-ESNWs, is an important subject in researchers interests to be counted as a technical work-in-progress.

Concluding Remarks
The trend in both fundamental and applied research on cellulose-and the cellulose-derivative ESNWs clearly indicates that this approach involves multidisciplinary science, which encompasses agriculture, engineering, and medicine. Including the related β(1,4)-type polysaccharides, chitin and chitosan, the scope for the practical applications of the ESNWs fabricated using natural polysaccharides and their derivatives will be extended with the increasing number of research publications, which have been growing in number remarkably since the latter half of the 1990s.
The relationships between basic polymer science and applied studies in ES technologies are complimentary to each other. Polymer science and the chemistry of cellulose have very long histories for both research and industries, thus, since the fabrication mechanism of nanofibers and ESNWs can also be understood and discussed, once the first cellulose-ESNW was developed and reported, the cellulose-ESNW technology immediately inspired researchers to hybridize it with the knowledge already accumulated in cellulose science. As a result, a variety of ideas summarized in Table 1 was developed within a very short period. There is a few number of examples in industrial applications of the cellulose-and cellulose-derivative ESNWs, however, due to the background mentioned above, the cellulosic ESNWs might have the most promising potential for industrialization. The recent progress in electrospinning technologies of chitin and chitosan [50][51][52] will also inspire/expand the potential applications of chemically modified (amino-functionalized) cellulose nanofibers [53][54][55].

Acknowledgments
This work was supported by Grants-in-Aid No. 26288101.

Conflicts of Interest
The author declares no conflict of interest.