Carbohydrate Polymers 132 (2015) 311–322

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Review

Review on biomedical and bioengineering applications of cellulose

sulfate
Qilei Zhang, Dongqiang Lin, Shanjing Yao ∗
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University,
Hangzhou 310027, China

a r t i c l e i n f o a b s t r a c t

Article history: Polysaccharide sulfates are naturally existing chemicals that show important biological activities in living
Received 30 April 2015 organisms. Cellulose sulfate is a semi-synthesized polysaccharide sulfate with a relatively simple chain
Received in revised form 11 June 2015 structure and unique biological properties and its biological applications have been explored in research
Accepted 12 June 2015
and clinical trials. With the advance of cellulose derivatization and characterization, cellulose sulfate
Available online 20 June 2015
molecules with tailored structures have been developed to fulfill individual requirements. This review
aims to provide a summary of recent development of cellulose sulfate in biomedical applications. Its
Keywords:
synthesis pathways were discussed with structure–property relationship elucidated. The application of
Cellulose sulfate
Cellulose sulfation
cellulose sulfate in drug delivery and microbe/cell immobilization were summarized with emphasis given
Antiviral on its polyelectrolyte complex formation processes.
Polyelectrolyte complexes © 2015 Elsevier Ltd. All rights reserved.
Cell immobilization

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
2. Cellulose sulfation: processing strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
2.1. Heterogeneous sulfation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
2.2. Homogeneous sulfation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
2.3. Quasi-homogeneous sulfation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
3. Structure–property relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
4. Biomedical applications of cellulose sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
4.1. Biological activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
4.1.1. Anticoagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
4.1.2. Antiviral: HIV treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
4.1.3. Cell regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
4.2. Polyelectrolyte complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
4.2.1. Drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
4.2.2. Microbe/cell immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
4.2.3. Mass transfer in PEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
5. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

∗ Corresponding author at: College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. Tel.: +86 571 87951982; fax: +86 571 87951982.
E-mail address: yaosj@zju.edu.cn (S. Yao).

http://dx.doi.org/10.1016/j.carbpol.2015.06.041
0144-8617/© 2015 Elsevier Ltd. All rights reserved.
312 Q. Zhang et al. / Carbohydrate Polymers 132 (2015) 311–322

1. Introduction shows the repeating units of heparan sulfate, chondroitin-6-sulfate

Polysaccharides are widely available biological polymers with
remarkable structural diversity. The unique backbone struc-
ture combined with good chemical amenability further makes
polysaccharides valuable resources. Currently, polysaccharides
find applications in almost every aspect of human life and with the
functionality of polysaccharide structures; it has become an impor-
tant material in textile, medicine, environmental protection, food
and pharmaceutical industry (Lucia & Habibi, 2012). Cellulose is a
simple polysaccharide with no branching or substituents, which is
often considered as the most abundant polymer in nature (Fox, Li,
Xu, & Edgar, 2011). It is massively produced with relatively low
cost and shows wide applications in both industry and academy
(Glasser, 2004).
The hydroxyl groups of cellulose can fully or partially react with
chemical agents to obtain various derivatives with different degree
of substitution (Habibi, 2014; Heinze & Petzold-Welcke, 2012), and
a series of cellulose derivatives like esters (El Seoud & Heinze, 2005)
and ethers (Nguyen, Devlin, Koshy, & Sorrell, 2014) have been syn-
thesized to meet specific requirements from industry. Table 1 listed
some typical cellulose derivatives currently available. Due to struc-
ture similarity and biological origin, these esters and ethers have
similar applications in food and pharmaceutical industry. Most of
these derivatives are also mass produced.
Cellulose sulfate is a semi-synthesized cellulose derivative with
a relatively simple chain structure and unique biological proper-
ties. Currently, it is still mainly produced in lab-scale with limited
commercial applications. Nevertheless, research has shown that
it can be applied as anticoagulant (Groth & Wagenknecht, 2001),
antiviral (McCarthy, 2007), contraceptive (Anderson et al., 2004),
human immunodeficiency virus (HIV) microbicide (Stone, 2002),
etc. These areas are hot spots for both academy and industry, and
the potential markets make cellulose sulfate unique among cel-
lulose derivatives. Similar biological activities of cellulose sulfate
were originally found on some naturally occurring polysaccharide
sulfates (Heinze, Daus, Gericke, & Liebert, 2010). For example, Fig. 1
and dermatan sulfate which are three important polysaccharide
sulfates found in living organisms. Meanwhile, cellulose sulfate
has certain advantages over natural counterparts, such as defined
chemical structure and easy separation/purification, which make it
suitable for industry-scale production and application.
This review would like to provide a summary of research on
cellulose sulfate, and emphasis is given to its application in biotech-
nological and medical areas, especially the application of cellulose
sulfate as a polyanionic polymer to form polyelectrolyte complex
films or capsules with polycations. Material synthesis pathways
are briefly reviewed and a future perspective is included to discuss
challenges and prospects of cellulose sulfate.
2. Cellulose sulfation: processing strategies
A series of synthetic procedures for the preparation of cellulose
sulfate have been investigated and methods for cellulose sulfation
were recently reviewed by Heinze et al. (2010, 2012). Therefore,
detailed synthetic methods and procedures will not be discussed
here. Generally, three synthetic strategies have been applied based
on the solubility of substrates in reaction media. Table 2 summa-
rized some typical synthetic methods reported in the literature.
2.1. Heterogeneous sulfation
Cellulose is difficult to be dissolved in most solvents (Pinkert,
Marsh, Pang, & Staiger, 2009). Therefore, if cellulose is used as
a raw material, sulfation process unfolds usually as a heteroge-
neous reaction. H2 SO4 and propanol are the most used reactants
for heterogeneous sulfation of cellulose (Yao, 2000), and recently
Chen, Zhang, Zhao, and Chen (2013) proposed a modified process
that used ethanol to replace propanol to simplify the synthetic
procedure and reduce cost. In order to restrain chain cleavage
catalyzed by H2 SO4 , the reaction is usually performed below
0 ◦ C. This restriction can be overcome by using SO3 complexes
such as SO3 -pyridine and SO3 -DMF as the sufation agents, and

Table 1
Examples of typical cellulose derivatives and their applications.

Cellulose derivatives Functional group Application References

Hydroxyethyl cellulose CH2 CH2 OH Thickener, pharmaceutical excipient Chronakis, Egermayer, and Piculell
(2002), Singh, Mishra, Singh, and
Narang (2003), Zulkifli, Hussain, Rasad,
and Mohd Yusoff (2014)
Hydroxypropyl cellulose CH2 CH(OH)CH3 Pharmaceutical excipient, lubricant, Nguyen and Latkany (2011), Ogawa
thickener et al. (2014)
Hydroxyethyl methyl cellulose CH2 CH2 OH, CH3 Thickener Mischnick et al. (2013)
Hydroxypropyl methyl cellulose CH2 CH(OH)CH3 , CH3 Pharmaceutical excipient, emulsifier, Jain et al. (2014), Khan, Anjum, Koya,
thickener and Kabir ud (2013), Lamberti et al.
(2013), Siepmann and Peppas (2012)
Ethyl hydroxyethyl cellulose CH2 CH2 OH, CH2 CH3 Emulsifier, pharmaceutical excipient Calejo et al. (2013), Karlberg,
Thuresson, and Lindman (2005)
Carboxymethyl cellulose CH2 COOH Thickener, food additive, Shlyapnikov, Shlyapnikova, and
pharmaceutical excipient Morozov (2014), Tang, Fan, Lin, and
Zhou (2014)
Methylcellulose CH3 Thickener, emulsifier Lott et al. (2013), Noronha, De
Carvalho, Lino, and Barreto (2014)
Ethylcellulose CH2 CH3 Emulsifier, pharmaceutical excipient Cheu, Chen, Chen, and Lin (2001),
Davidovich-Pinhas, Barbut, and
Marangoni (2015)
Cellulose acetate COCH3 Coating, fiber Caruso and Schattka (2000), Liu and
Hsieh (2002)
Cellulose propionate COCH2 CH3 Coating, film Bianchi, Marsano, Ricco, and Conio
(1997)
Cellulose acetate propionate COCH2 CH3 , COCH3 Fiber, paint Huang et al. (2011)
Cellulose acetate butyrate COCH2 CH2 CH3 , COCH3 Fiber, paint Huang et al. (2011)
Cellulose nitrate NO2 Fiber, film, explosive Nartker and Drzal (2010)
Cellulose sulfate SO3 H Microbicide, anticoagulant, Aggarwal et al. (2013), Neurath (2008),
pharmaceutical excipient, film Wang et al. (1997)
 Q. Zhang et al. / Carbohydrate Polymers 132 (2015) 311–322 313

Fig. 1. Repeating units of some naturally occurring polysaccharide sulfates and cellulose sulfate.

dimethylformamide (DMF) is usually used as the solvent to suspend
cellulose during reaction (Zhu et al., 2014). Since cellulose sulfate
has to reach certain substitution degree to be dissolved in DMF,
this method often results in products with high degree of substitu-
tion. The heterogeneous strategy uses simple raw materials with
flexible preparation procedures. However, one problem for het-
erogeneous synthesis is non-uniform substitution. As cellulose is
not fully dissolved, substitution mainly happens at the amorphous
parts of cellulose with the crystalline part remain unreacted.
2.2. Homogeneous sulfation
(BMIMCl), 1-ethyl-3-methylimidazolium acetate (EMIMAc) and
1-allyl-3methylimidazolium chloride (AMIMCl) in homogeneous
synthesis of cellulose sulfate. The results showed that direct sul-
fation under completely homogeneous conditions was applicable.
The minimum degree of substitution required for water solubility
of the product was 0.25. It is worth to note that the cellulose-ionic
liquid system usually has high viscosity which sometimes makes
mixing problematic. Therefore, cosolvents are usually needed to
reduce the system viscosity. Synthesis in ionic liquids seems a good
pathway to prepare cellulose sulfate, but the high cost of ionic
liquids needs to be aware when considering mass production.

Homogeneous strategy can overcome this non-uniform sub-
stitution problem after dissolving cellulose in certain solvents.
Products from homogeneous synthesis usually have even distri-
bution, good solubility and low chain degradation (Qin et al.,
2014). For example, N2 O4 /DMF was used for the synthesis of cel-
lulose sulfate with SO3 -complexes as reactants (Wagenknecht,
Nehls, & Philipp, 1993). Cellulose nitrite is first formed as an
intermediate. It then reacted with SO3 complexes to obtain final
products with different degree of substitution. Moreover, cellu-
lose sulfate synthesized can be dissolved in water at very low
degree of substitution (∼0.3). However, N2 O4 is toxic which lim-
its large-scale production and also the application of the product
in biomedical areas (Gericke, Liebert, & Heinze, 2009a). Mean-
while, ionic liquids have become extremely popular in research
(Rogers & Seddon, 2003) and some are good dissolution media for
cellulose (Gericke, Liebert, & Heinze, 2009b; Pinkert et al., 2009).
Gericke et al. (2009a) used 1-butyl-3-methylimidazolium chloride
2.3. Quasi-homogeneous sulfation
In addition to homogeneous synthesis, cellulose derivatives
with better solubility than cellulose were used as reactants. Such
strategies are referred as quasi-homogeneous synthesis and the
properties of cellulose sulfate prepared are usually dependent
on sulfation processes. Cellulose acetate, cellulose nitrite and
trimethylsilyl cellulose (TMS) are three typical derivatives used and
the main problem of this strategy is the complexity of reaction sys-
tems which need further effort for purification (Richter & Klemm,
2003).
The above-discussed methods for cellulose sulfate synthesis are
mainly for the preparation of sodium salts. The development of
sulfated cellulose has been further explored in studying deriva-
tives of cellulose sulfate (Heinze & Rahn, 1999). For example,
methylated cellulose sulfate (Gohdes, Mischnick, & Wagenknecht,
1997), carboxyl cellulose sulfate (Zhang et al., 2010b), amino

Table 2
Typical pathways for cellulose sulfate synthesis.

Synthesis Typical chemical Characteristics Reference

Heterogeneous H2 SO4 /propanol or ethanol Low temperature is necessary,
non-uniformly substituted, cheap raw
material and good adjustability
SO3 -complex* /DMF High degree of substitution,
non-uniformly substituted
Quasi-homogeneous Cellulose acetate Good reactant solubility, need further
purification
Trimethylsilyl (TMS) cellulose Degree of substitution is limited by
TMS, negligible depolymerization
SO3 -complex/acetic anhydride Regioselective synthesis, good water
solubility, need large amount of
chemicals
Homogeneous N2 O4 /DMF Good reactant and product solubility,
high solvent toxicity
Ionic liquid Good reactant and product solubility,
uniform substitution, high viscosity
*
SO3 complex can be SO3 -DMF, SO3 -pyridine, ClSO3 H, etc.
Bhatt, Gupta, and Naithani (2008),
Chen et al. (2013), Yao (2000)
Zhu et al. (2014)
Zhang, Peschel, Brenaler, Groth, and
Fischer (2009)
Richter and Klemm (2003)
Zhang et al. (2011a), Zhang, Peschel,
Bäucker, Groth, and Fischer (2011b)
Wagenknecht et al. (1993)
Gericke et al. (2009a), Liebert et al.
(2009), Tao, Song, and Chou (2011)
314 Q. Zhang et al. / Carbohydrate Polymers 132 (2015) 311–322
cellulose sulfate (Heinze, Genco, Petzold-Welcke, & Wondraczek,
2012) and oxyethylated cellulose sulfate (Udoratina, Torlopov,
Drozd, & Makarov, 2012) were synthesized with their biological
properties compared.
With the development of modern chemistry, fine manipu-
lation of molecule becomes possible and sometimes needed to
obtain chemicals with specific properties. Reaction conditions are
milder when cellulose is fully dissolved that breaks extensive H-
bonds, which make it possible for regioselective and regiospecific
synthesis of cellulose derivatives (Fox et al., 2011). The regioselec-
tive synthesis of cellulose sulfate has been reviewed (Baumann,
Liu, & Faust, 2003; Fox et al., 2011; Klemm, Heinze, Philipp, &
Wagenknecht, 1997), and heterogeneous, quasi-homogeneous and
homogeneous strategies have all been applied with various reac-
tants and solvent systems. However, many researchers only use 1 H
and 13 C NMR spectra data to support the regioselective synthesis,
which are not always convincing. Therefore, it is also important to
develop a comprehensive characterizing package for such research
(Gohdes & Mischnick, 1998).
3. Structure–property relationship
Like many other polymers, the physical, chemical and biologi-
cal properties of cellulose sulfate, such as water solubility, chemical
reaction and biological activity, are largely dependent on the molec-
ular weight, the degree and distribution of substitution along poly-
mer chains (Heinze et al., 2010). It has been noted that the quantifi-
cation of cellulose sulfation remains challenging (Gu, Catchmark,
Kaiser, & Archibald, 2013). Several characterizing techniques
have been applied in structure study. For example, Raman and
infrared spectroscopy were used to measure substitution degree
(Petropavlovskii & Vasil’eva, 1971; Zhang, Brendler, & Fischer,
2010a). The correlation between certain Raman vibration signals
with total degree of substitution was built and the correlation
results were compared with 13 C nuclear magnetic resonance (NMR)
spectroscopy and elemental analysis results, which indicated that
Raman spectroscopy can be a potential rapid method for quanti-
fying degree of substitution (Zhang, Brendler, Geissler, & Fischer,
2011a). Zhu and Yao (2012) compared polyelectrolyte titration,
BaSO4 turbidimetry and elemental analysis methods in the mea-
surement of substitution degree. They found that BaSO4 turbidime-
try and elemental analysis can provide similar results, but the poly-
electrolyte titration lacked consistency. Moreover, the distribution
of sulfate group is an important physical factor that can affect
water solubility of cellulose sulfate. Gohdes and Mischnick (1998)
used a series of methods including permethylation, desulfation,
deuteromethylation, random cleavage, remethylation and fast-
atom bombardment mass spectrum to determine the substitution
pattern of cellulose sulfate by analysis of degraded samples. Cellu-
lose sulfate prepared from seven different sulfation processes were
compared. Their work provided detailed results about positions of
sulfated groups on cellulose chains. Two-dimensional 1 H and 13 C
NMR spectroscopy (Kowsaka, Okajima, & Kamide, 1991) and mass
spectrometry (Mischnick, Heinrich, & Gohdes, 1999) were also
applied in the determination of substitution distribution. Moreover,
the molecular weight of cellulose sulfate can be measured by low
angle laser light scattering (LALLS) and gel permeation chromatog-
raphy (GPC) (Wang & Yao, 2009; Zhu & Yao, 2012), while LALLS has
advantages of measurement without standard polymer samples.
Other properties of cellulose sulfate such as liquid crystal formation
were also discussed in literature studied by differential scanning
calorimetry (DSC) and X-ray diffractometry (Hatakeyama, Yoshida,
& Hatakeyama, 1995; Onishi, Hatakeyama, & Hatakeyama, 2009).
The performance of polymer materials in various applications
is usually closely related to their structures. Table 2 shows that
Fig. 2. Relationship of viscosity, degree of substitution and soluble part percentage
of cellulose sulfate synthesis by heterogeneous pathways.
the structure properties are most likely determined by the reac-
tion pathways. Heterogeneous synthesis usually results in the
formation of non-uniform substitution distribution, which is due
to the solubility difference of amorphous and crystalline parts
of cellulose in solvents. Meanwhile, the degree of substitution is
affected by reaction type and time. For examples, the relation-
ship between degree of substitution, percentage of water soluble
parts and viscosity was studied under heterogeneous synthesis
with H2 SO4 /propanol mixtures and the results can be schemati-
cally depicted (see Fig. 2) (Heinze & Petzold-Welcke, 2012). It seems
reasonable that with the increase in soluble part percentage and
degree of substitution, the viscosity of cellulose sulfate decreases.
The effect of sulfate groups on the properties of cellulose sul-
fate has been widely studied, especially on sulfated nanocrystalline
cellulose. For example, Voronova, Surov, and Zakharov (2013) pre-
pared films composed of nanocrystalline cellulose sulfate and the
relationship between film property and sulfate group contents
was studied. The results showed that both surface roughness and
water adsorption ability enhanced with the increase in sulfate
group. Jiang, Kittle, Tan, Esker, and Roman (2013) used sulfated
cellulose as substrates for cellulase to study the effects of sul-
fate group on cellulase adsorption and enzymatic hydrolysis. They
found that the surface accessibility was similar with different sul-
fate group density, but the binding domain decreased. An inhibitory
effect of sulfate group in enzymatic hydrolysis was discussed.
Moreover, the effects of sulfate groups on the thermal degrada-
tion of cellulose were studied (Lin & Dufresne, 2014; Roman &
Winter, 2004). The results showed that the degradation temper-
ature was significantly decreased with the sulfation of cellulose,
and the sulfate group showed a catalytic effect on cellulose ther-
mal degradation. Lokanathan, Uddin, Rojas, and Laine (2013) used
cellulose nanocrystals to assist the synthesis of silver nanoparti-
cles. The results showed that cellulose after sulfation can stabilize
silver nanoparticles formation and the decrease in substitution
resulted in narrower nanoparticle size distribution. Moreover, cel-
lulose sulfate with high degree of substitution has shown potential
application as microbicides (Anderson et al., 2002; Yoshida, 2001;
Yoshida et al., 2001).
4. Biomedical applications of cellulose sulfate
4.1. Biological activity
Polysaccharide sulfates in living organisms were early found
playing certain roles in their biological activities, such as antivi-
ral and blood coagulation (Ghosh et al., 2009; Kindness, Long, &
 Q. Zhang et al. / Carbohydrate Polymers 132 (2015) 311–322
Table 3
Typical biological activities and applications of cellulose sulfate.
Biological activity Application
Anticoagulation Anticoagulation
Anticoagulant coatings
Antiviral Papillomavirus microbicide
Vaginal microbicide
Human immunodeficiency virus (HIV) treatment
Contraception
Cell regulation Cell expression/proliferation regulation
Williamson, 1980). Currently, various semi-synthesized polysac-
charide sulfates have been developed and similar biological
behaviors are also found from these materials, such as chitosan sul-
fate, dextran sulfate and cellulose sulfate (Groth & Wagenknecht,
2001). This review is focused on the application of cellulose sul-
fate with some typical biological activities and related applications
summarized in Table 3.
4.1.1. Anticoagulation
Natural heparin is a widely used anticoagulant in clinical appli-
cation but shows certain side effects (Han, Yao, Yang, Liu, & Gao,
2005). Therefore, development of alternatives like cellulose sulfate
is important. Moreover, cellulose sulfate has well-defined struc-
ture and lack of branches on polymer chains, which is good for
quality control and structure–activity research. Wang, Li, Zheng,
Normakhamatov, and Guo (2007) found that cellulose sulfate
showed higher anticoagulation activity than heparin under their
experimental conditions, and in vivo studies revealed that the
anticoagulation activity of cellulose sulfate was achieved by accel-
erating the inhabitation of antithrombin III on coagulation factors
in plasma. Similar anticoagulation mechanism was discussed by
Grombe et al. (2007). The effects of molecular weight and sul-
fate group distribution on anticoagulation were also investigated
(Wang, Li, Chen, Li, & Guo, 2005; Wang, Xiao, Li, & Wu, 2010c).
Although it was revealed that molecular weight was important in
the anticoagulation application, their in vitro and in vivo results
seem controversial and further research may be necessary in study-
ing the structure–activity relationship.
4.1.2. Antiviral: HIV treatments
Antiviral has a large market in pharmaceutical industry and is a
major focus for biotech companies (McCarthy, 2007). Among newly
developed antiviral substances in recent years, sulfated polysac-
charides are often highlighted. Some aspects of their antiviral
activity have also been elucidated, which becomes clear that the
charge density and chain length are not the only contributors in
their antiviral performance (Ghosh et al., 2009). Cellulose sulfate
is one of the most reported sulfated polysaccharides in antiviral
investigation, which may mainly be attributed to a commercial
drug candidate named UshercellTM . It is a high-molecular-weight
form of cellulose sulfate developed by Polydex (Canda) and was
found active against human immunodeficiency virus (HIV), herpes
simplex virus (HSV), Neisseria gonorrhoeae, Chlamydia trachomatis,
papillomaviruses and gardnerella vaginalis (Anderson et al., 2004).
UshercellTM also shows contraceptive in preclinical evaluations
(Anderson et al., 2002) and it has excellent safety performance
in different studies (El-Sadr et al., 2006; Mauck, Frezieres, Walsh,
Robergeau, & Callahan, 2001a; van der Straten et al., 2007) and
phase I evaluation (Mauck et al., 2001b). However, phase III study
with a total of 1398 women enrolled concluded that cellulose sul-
fate did not prevent HIV infection and may increase the risk of
HIV acquisition (Van Damme et al., 2008). The lack of consistence
between phase III and pre-phase III studies is still under discus-
sion (Check, 2007; Neurath, 2008) and it is worth to re-evaluate
the clinical results to solve related controversy.
315
Reference
Wang et al. (2007), Wang et al. (2010c)
Gericke et al. (2011)
Christensen et al. (2001)
Simoes et al. (2002)
El-Sadr et al. (2006), McGowan (2006), Stone (2002)
Mauck et al. (2001a, 2008), van der Straten et al. (2007)
Aggarwal et al. (2013, 2014), Morioke et al. (2012), Peschel et al. (2010)
4.1.3. Cell regulation
Cell growth and stem-cell differentiation are regulated by var-
ious factors such as growth factors and extracellular matrices.
The binding between sulfated cellulose and fibroblast growth
factors (FGF) have been studied in the literature. For example,
Weltrowski et al. (2012) studied the mitogenic activity of cellu-
lose sulfate and its protective effect against proteolytic digestion
of FGF-2. Their results indicated that the mitogenic activity of
cellulose sulfate was related to the substitution degree and con-
centration of the polymer, which showed an increase in cell
growth with the increase substitution degree and concentration.
Moreover, they found that the mechanism on mitogenic activity
was mainly related to a prolonged life-time of FGF-2 by protec-
tion of FGF-2 against proteolytic digestion. Peschel et al. (2010)
investigated the cytotoxicity and motigenic activity of cellulose
sulfate by modulation of 3T3 fibroblast proliferation with or with-
out FGF-2. Their results showed that cellulose sulfate and their
carboxyl and carboxymethyl derivatives were all non-toxic to
3T3 cells. Cellulose sulfate strongly promoted cell proliferation
by FGF-2 induction and this promotion was substitution degree-
related. Moreover, cellulose sulfate is better than heparin in the
absence of FGF-2. Meanwhile, no significant promoting effects
were found with carboxyl and carboxymethyl derivatives of cel-
lulose sulfate. In addition, the suppression of immunoglobulin E
expression by cellulose sulfate was studied (Morioke et al., 2012)
and the application of cellulose sulfate pairing with other poly-
electrolytes on cell culture will be discussed in the following
section.
4.2. Polyelectrolyte complexes
Polyelectrolyte complexes (PEC) usually refers to polymer sys-
tems formed by mixing oppositely charged polyelectrolytes in
solutions without covalent crosslinkers (Luo & Wang, 2014). The
formation process is based on the interaction between polyionic
polymers via electrostatic and dipole–dipole association, hydrogen
bonds and hydrophobic interactions. Therefore, the stability of PEC
can be affected by various factors, such as ionic strength and pH.
Due to mild reaction conditions and wide selection of polymers,
PEC is often studied for drug delivery and biological applications
(Bertin, 2014; Müller, 2014).
Cellulose sulfate is a semi-synthesized polyanionic polymer that
can form PEC with various natural or synthetic polycations, such
as chitosan and poly-dimethyl-diallyl-ammonium chloride. More-
over, the unique biological properties of cellulose sulfate discussed
in this review make it suitable and sometimes advantageous for
some high-demanding biotechnology applications, such as drug
delivery and cell immobilization. Table 4 summarized cellulose
sulfate-related PEC reported in the literature and Fig. 3 shows the
chemical structures of the materials listed in Table 4. Microen-
capsulation (Bhujbal, de Vos, & Niclou, 2014) and layer-by-layer
techniques (De Villiers, Otto, Strydom, & Lvov, 2011) are two
of the most widely used approaches for cellulose sulfate PEC
preparation.
316 Q. Zhang et al. / Carbohydrate Polymers 132 (2015) 311–322

Table 4
Typical cellulose sulfate-related polyelectrolyte complexes (PEC) studied in the literature.

Chemical Application Preparation References

Alginate/poly(methylene-co- Microbe/cell immobilization Sodium alginate and cellulose sulfate solutions
guanidine) were mixed and dropped into CaCl2 and PMCG
(PMCG) solution
A two-step process for preparing
alginate-cellulose sulfate-PMCG microcapsules
A new device for microcapsule preparation
with controlled membrane and size
Alginate and cellulose sulfate were mixed and
gelled in calcium chloride solution
Briššová, Lacík, Powers, Anilkumar, and
Wang (1997), Bučko et al. (2005, 2006),
Canaple, Rehor, and Hunkeler (2002),
Lacík, Briššová, Anilkumar, Powers, and
Wang (1997), Müller et al. (1999),
Nahalka et al. (2008), Podskočová,
Chorvát, Kolláriková, and Lacík (2005),
Rehor, Canaple, Zhang, and Hunkeler
(2001), Renken and Hunkeler (2007),
Vikartovská et al. (2007), Wang et al.
(1997), Zhang et al. (2003), Zhang, Yao,
and Guan (2005b)
Lacík, Anilkumar, and Wang (2001)
Ceausoglu and Hunkeler (2002)
Ponce, Orive, Gascón, Hernández, and
Pedraz (2005)

Chitosan Cell culturing Chitosan and cellulose sulfate films were
prepared via a layer-by-layer method
Drug delivery Chitosan and cellulose sulfate films were
prepared via a layer-by-layer method
Chitosan and cellulose sulfate solutions were
mixed
Cellulose sulfate was dropped into chitosan
solution and crosslinked with tripolyphosphate
Aggarwal et al. (2013, 2014)
Xie et al. (2009)
Wang et al. (2009, 2010a), Zhu, Lin, and
Yao (2010)
Wu and Yao (2013), Zhang et al. (2013)

Water soluble chitosan Drug delivery Cellulose sulfate was dropped into water Wu et al. (2013, 2014)
soluble chitosan solution and crosslinked with
polyphosphate

Poly-dimethyl-diallyl- Microbe/cell immobilization PDMDAAC and cellulose sulfate were
ammonium chloride deposited layer-by-layer on CaCO3 surface to
(PDMDAAC) obtain hollow microcapsules
Cellulose sulfate solution was dropped into
PDMDAAC solution
Starch was added in the system and later
removed by amylase to make macroporous
capsules
Alginate was added in the system and later
removed by dissolution
Carboxymethyl cellulose was added in the
cellulose sulfate/PDMDAAC system to adjust
microcapsule properties
Tan, Ren, and Yao (2011), Yao, Guan,
and Lin (2006)
Dautzenberg et al. (1999a, 1999b),
Fluri et al. (2008), Groot-Wassink et al.
(1992), Liu et al. (2010), Mansfeld et al.
(1991, 1995), Merten et al. (1991),
Samel et al. (2006), Son et al. (2004),
Stadlbauer et al. (2006), Štefuca et al.
(1991), Weber et al. (2006), Zeng et al.
(2012, 2013a), Zhang et al. (1999),
Zhang, Guan, Ji, and Yao (2006)
Zhang et al. (2005a)
Weber et al. (2004)
Chen, Yao, Guan, and Lin (2005)

Poly(ethyleneimine) Drug delivery, microbe/cell Poly(ethyleneimine) was mixed with cellulose Müller and Keßler (2012), Woltmann
immobilization sulfate under stirring et al. (2014)

Hexadecylpyridinium chloride Membrane separation Layer-by-layer formation Tamaddondar et al. (2014)

(surfactant)
Dip-coating process Schwarz et al. (2001)

4.2.1. Drug delivery
The efficiency of drug used by patients is often limited by
delivery route, drug instability, immunogenicity and physiolog-
ical barriers. Therefore, tremendous efforts have been made in
developing drug-delivery systems (Lu, Sun, & Gu, 2014). Tech-
niques such as extrusion, emulsion and spray drying are often
used in industry for the production of drug particles encapsu-
lated with various materials (Cook, Tzortzis, Charalampopoulos,
& Khutoryanskiy, 2012). However, drugs-like proteins have spe-
cial preparation requirements, and mild operation conditions are
often needed. PEC microencapsulation is a suitable approach for
such applications, and literatures show that polysaccharides such
as alginate, chitosan and cellulose derivatives are the most common
encapsulation agents due to their biocompatibility, easy availability
and unique biological properties (Cook et al., 2012).
Research found that cellulose sulfate synthesized via homo-
geneous pathway is preferred for microencapsulation, because
good water solubility, high viscosity and uniform sulfate group
distribution are favored in PEC formation (Gericke et al., 2009a).
Hartlig, Carlesso, Davidson, and Prokop (2007) studied the effects of
molecular weight of cellulose sulfate on related PEC physicochemi-
cal properties. Sodium alginate and poly(methylene-co-guanidine)
(PMCG) were used as coupling polymers and the results showed
that polymers with similar low molecular weights resulted in
PEC dispersions with suitable physicochemical properties, while
significantly different molecular weight polymers lead to water
 Q. Zhang et al. / Carbohydrate Polymers 132 (2015) 311–322
Fig. 3. Structures of chemicals used in the preparation of polyelectrolyte complexes (PEC) with cellulose sulfate.
insoluble aggregates. Müller and Keßler (2012) used a solution
casting method to prepare poly(ethyleneimine)/cellulose sulfate
PEC films as controlled release matrices for pamidronate, and the
results showed that the drug release kinetics can be adjusted by
changing polymer mixing ratios. Supercritical fluid-assisted atom-
ization was also applied in the preparation of cellulose sulfate,
and spherical particles with narrow size distribution and mean
diameter from 0.3 to 3.0 ␮m can be obtained (Wang, Guan, Yao,
& Zhu, 2010b). In addition, chitosan as a polycation is often used
with cellulose sulfate to form PEC microcapsules. For example, 5-
aminosalucylic acid and lactoferrin were encapsulated individually
with chitosan/cellulose sulfate/polyphosphate and good encapsu-
lation ratio and designed drug release profiles can be obtained
(Wu, Li, & Yao, 2014; Wu & Yao, 2013; Wu, Zhang, Lin, & Yao,
2013). Moreover, research showed the chitosan–cellulose sulfate
system can be used as a potential microbial-trigged colon-specific
drug-delivery system (Wang, Xie, Chen, & Yao, 2010a), as the PEC
capsules formed by chitosan–cellulose sulfate can only be degraded
in the colon region after oral intake due to the rich of microbial in
human colon. For example, Fig. 4 shows two blank capsules pre-
pared by chitosan–cellulose sulfate PEC and the actual size can be
adjusted accordingly (Wang, Xie, Zheng, & Yao, 2009).
Layer-by-layer assembly is a more delicate technique for the
encapsulation of drugs and living organisms with cellulose sulfate
(Aggarwal & Groth, 2014). It can also be used as surface coatings
to modify various material surfaces (Baumann et al., 2003; Müller,
Briššová, Rieser, Powers, & Lunkwitz, 1999). Multilayer PEC films
or capsules are usually prepared by a dipping–washing–dipping
process, i.e. dip the template in one of the polymer solutions for
a certain time interval and then wash with distilled water and
dip into the pairing polymer solution. The whole process can be
repeated until desired films are achieved. Fig. 5 is a schematic
diagram showing the layered structure of chitosan and cellulose
Fig. 4. Blank capsules prepared with chitosan and cellulose sulfate. Adapted with
permission from Wang et al. (2009). Copyright (2009) American Chemical Society.
317
sulfate (Xie, Wang, & Yao, 2009). A detailed PEC forming mech-
anism was discussed which shows the existence of competitive
interaction between polyelectrolytes (Zhang, Wu, Lin, & Yao, 2013).
4.2.2. Microbe/cell immobilization
Cell immobilization or encapsulation is used to protect cells
from external environment and provide microenvironment for
cell promotion, and controlling cell viability, proliferation and
release of therapeutic chemicals (Acarregui, Murua, Pedraz, Orive,
& Hernndez, 2012; Orive et al., 2004; Orive, Santos, Pedraz, &
Hernández, 2014). This technique is often considered for disease
treatments, which can be treated as advanced drug-delivery sys-
tems with living cells as individual bio-factories. Therefore, it is
important that the encapsulating materials do not have negative
effects on cell growth, and it would be advantageous if the mate-
rials used can facilitate drug production of living cells. Cellulose
sulfate has unique biological activities and research has shown
its potential applications in cell regulation (Table 3). Therefore,
it is a promising material for cell immobilization/encapsulation.
Cells such as antibody expressing mammalian cells (Dautzenberg
et al., 1999a), cellulase secreting mammalian sensor cells (Fluri,
Kemmer, Daoud-El Baba, & Fussenegger, 2008), jurkat cells (Kaiser
et al., 2014; Werner et al., 2013), hybridoma cells (Shen, Reid,
& Greenfield, 1993), yeast cells (Zhang, Mei, & Yao, 1999), co-
immobilized Yarrowia lipolytica cells and invertase (Mansfeld,
Förster, Hoffmann, Schellenberger, & Dautzenberg, 1995), human
mesenchymal stromal cells (Woltmann, Torger, Müller, & Hempel,
2014), nocardia tartaricans cells (Bučko et al., 2006), Chinese ham-
ster ovary cells (Weber et al., 2004) and Choristoneura funiferana
cells (Son et al., 2004) have all been immobilized using cellulose
sulfate and in vitro and in vivo studies were performed. Some of
the immobilization methods and materials are listed in Table 4.
Cellulose sulfate paired with poly-dimethyl-diallyl-ammonium
chloride (PDMDAAC) is the most popular PEC forming combination
(Merten, Dautzenberg, & Palfi, 1991), which has advantages of sta-
ble physiochemical properties, robust mechanical strength, good
biocompatibility and sharp cut-off membranes. A typical encap-
sulation process with cellulose and PDMDAAC is illustrated in
Fig. 6.
A cytochrome P450 enzyme expressing and genetically modi-
fied allogeneic cells were immobilized in cellulose sulfate capsules
and its effects in pancreatic cancer treatments were evaluation
in a phase I/II trial (Löhr et al., 2001). The authors concluded
that higher survival rates might be obtainable with this strategy
than another cytotoxic agent. Cellulose sulfate-related PEC has
also been studied in detailed for pancreatic islet cell transplan-
tation for the treatment of diabetes mellitus (Stadlbauer et al.,
2006). Wang et al. (1997) screened over a thousand combina-
tions of polyanions and polycations to search for new polymer
318 Q. Zhang et al. / Carbohydrate Polymers 132 (2015) 311–322
Fig. 5. Schematic diagram of the layer-by-layer assembly with chitosan and cellulose sulfate. Adapted with permission from Xie et al. (2009). Copyright (2009) American
Chemical Society.
candidates that may suitable for encapsulating pancreatic islets.
They found that the combination of sodium alginate, cellulose
sulfate, PMCG and some chloride salts was the most promis-
ing. Stiegler et al. (Schaffellner et al., 2005; Stiegler et al., 2006)
encapsulated an insulin-producing cell line HIT-T15 with cellu-
lose sulfate and PDMDAAC and they demonstrated that cellulose
sulfate is less immunogenic and more biocompatible than other
Fig. 6. Schematic diagram of biological material encapsulation with polyelectrolyte
complexes formed by cellulose sulfate and poly-dimethyl-diallyl-ammonium chlo-
ride (PDMDAAC). Adapted with permission from Gericke et al. (2009a). Copyright
(2009) WILEY-VCH Verlag GmbH & Co.
materials, and it did not influence cell growth and insulin produc-
tion. Essential nutrients and insulin can pass through the capsule
membrane without significant delay. Moreover, it was also found
that freezing and thawing of the cells immobilized in cellulose sul-
fate is possible.
In addition, the advantages of cellulose sulfate in cell encapsu-
lation made it also a promising candidate for microbe and enzyme
immobilization. For example, bacteria such as Klebsiella pneumo-
niae was encapsulated in cellulose sulfate-PDMDAAC microcap-
sules for the production of 1,3-propanediol under both batch and
continuous modes (Zhao, Chen, & Yao, 2006). The results showed
that the biomass concentration was 2.6 times higher than that of
free culturing in the microcapsules, and the substrate tolerance and
microbe activity were both increased. Lactate dehydrogenase was
also encapsulated in PEC matrices formed by dropping 2% (w/w)
cellulose sulfate solution into 2% (w/w) PDMDAAC solution (Štefuca
et al., 1991). The results indicated that this mild preparation con-
dition was suitable for sensitive enzymes. Moreover, the enzyme
can move freely in PEC without leakage, while the substrates can
easily penetrate into the semipermeable microcapsules. Nahalka,
Dib, and Nidetzky (2008) studied the immobilization of d-amino
acid oxidase in alginate/PMCG/cellulose sulfate/CaCl2 systems and
approximately 52% immobilization yield (expressed as the fraction
of original activity) was achieved. The enzyme entrapped was 720-
fold more stable than free enzymes, which indicates the potential
industry value of this technique. The same PEC system was used
in encapsulation of glucose oxidase by Vikartovská et al. (2007)
and the results showed that the encapsulated enzyme had higher
thermostability and storage stability. The reusability was also dra-
matically enhanced compared to free enzymes. Zeng et al. (2012,
2013a, 2013b) used the cellulose sulfate–PDMDAAC system to
immobilize microalgae Chlorella sp. for removing total nitrogen and
phosphate in wastewater. They found that the capsules prepared
 Q. Zhang et al. / Carbohydrate Polymers 132 (2015) 311–322
had good mechanical stability, biocompatibility and high substrate
tolerance. Moreover, the microalgae immobilized had a robust
morphology. Furthermore, the cellulose sulfate–PDMDAAC system
have also been applied in other microbe/enzyme investigations,
such as monascus purpureus for natural pigment production (Liu,
Ren, & Yao, 2010), invertase (Mansfeld, Förster, Schellengberger,
& Dautzenberg, 1991), bovine spermatozoa (Weber, Rimann,
Schafroth, Witschi, & Fussenegger, 2006) and brevibacterium
flavum (Mei, Yang, Zhong, Lin, & Yao, 2005).
4.2.3. Mass transfer in PEC
The physical, chemical and biological properties of encapsu-
lating materials are the basis in determining the applicability of
living organism immobilization. However, mass transfer inside
those semi-permeable matrices/membranes is often vital in deter-
mining the success of such applications, as nutrients need to be
transferred in and products need to be transferred out. For example,
the diffusion coefficient of glucose in cellulose sulfate–PDMDAAC
membranes was investigated with a diffusion model and was found
at ∼2.12 × 10−11 m2 /s (Shanjing & Man-Gi, 1998). Groot-Wassink,
Dautzenberg, Grunow, and Von Baehr (1992) used cellulose sul-
fate and PDMDAAC for hybridoma cell encapsulation and found
that albumin and transferrin were not able to diffuse through the
membrane, although the system protected cells from damage. One
advantage of PEC systems is that the diffusion characteristics can be
adjusted by changing preparation procedures and processes. Zhang,
Yao, and Guan (2003) prepared a series of alginate/PMCG/cellulose
sulfate systems with different polymer concentrations and the
diffusivity of nine solutes was measured to establish diffusion
mathematical models. The effect of molecular weight of PDMDAAC
on the cellulose sulfate–PDMDAAC membrane permeability was
studied by Dautzenberg, Schuldt, Lerche, Woehlecke, and Ehwald
(1999b). The results showed that a homogeneous membrane pore
structure and a sharp membrane cut-off of ∼2 nm can be obtained
with PDMDAAC molecular weight >10 kDa, which is enough for sta-
ble inclusion of enzymes with molecular weight >20 kDa. In order to
prepare membranes with higher-molecular-weight cut-off, macro-
porous cellulose sulfate–PDMDAAC membranes were prepared by
adding starch in the system which was later hydrolyzed by amylase
(Zhang, Yao, & Guan, 2005a). The molecular cut-off of the mem-
brane was 70 kDa for globular proteins that was approximately five
times larger than systems without starch. Moreover, membranes
prepared from cellulose sulfate-related PEC were also applied in
membrane separation of gas mixtures (Schwarz, Apostel, & Paul,
2001; Tamaddondar, Pahlavanzadeh, Saeid Hosseini, Ruan, & Tan,
2014).
5. Conclusions and future perspectives
Cellulose sulfate is a cellulose derivative with unique biologi-
cal properties. Its bioactive and ionic nature made it a promising
candidate for live organism immobilization and even can be used
as pharmaceutical active ingredients in disease treatments. This
review presented a summary of current research on cellulose sul-
fate. The synthesis methods were discussed and the emphasis was
focused on its application in biotechnological fields. Nevertheless,
cellulose sulfate is still a lab product without commercialization.
The materials used in research are most individually prepared.
Therefore, results from different groups are not comparable and
sometimes lack consistence. It is important to develop affordable
and robust synthesis pathways to obtain products with reliable
quality, and also explore techniques and methods for clarifying
their chemical structures. Furthermore, regioselective synthesis of
cellulose sulfate is vital in fulfilling high-demanding requirements
from biological industry.
319
Acknowledgements
This work was financially supported by the National Natural Sci-
ence Foundation of China and the Doctoral Programs Foundation of
Ministry of Education of China (No. 20110101130007).
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