A low-toxic artificial fluorescent glycoprotein can serve as an efficient cytoplasmic labeling in living cell

Article abstract of DOI:10.1016/j.carbpol.2014.08.118

To maintain the virtue of good optical property and discard the dross of conventional fluorescent staining dyes, we provide a strategy for designing new fluorescent scaffolds. In this study, a novel fluorescent labeling glycoprotein (chitosan-poly-l-cysteine, CPC) was synthesized through graft copolymerization. CPC gives emission peak at 465470 nm when excited at 386 nm. The submicro-scale CPC microspheres could be localized and persisted specifically in the cytoplasm of living cells, with strong blue fluorescence. Moreover, CPC was highly resistant to photo bleaching, the fluorescence was remained stable for up to 72 h as the cells grew and developed. The glycoprotein CPC was bio-compatible and in zero grade cytotoxicity as quantified by MTT assay. The fluorescent labeling process with our newly designed glycoprotein CPC is exceptionally efficient.

Full text of DOI:10.1016/j.carbpol.2014.08.118

Carbohydrate Polymers 117 (2015) 211–214
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Short communication
A low-toxic artificial fluorescent glycoprotein can serve as an efficient
cytoplasmic labeling in living cell
Jiangju Sia,b, Dawei Lianga,b, Dan Kongc, Sufang Wua,b, Lan Yuand, Yan Xianga,b,∗
,
Lei Jianga,b
a Key Laboratory of Bio-Inspired Smart Interfacial Science and Technology of Ministry of Education, School of Chemistry and Environment, Beihang
University, Beijing 100191, PR China
b Beijing Key Laboratory of Bio-Inspired Energy Materials and Devices, School of Chemistry and Environment, Beihang University, Beijing 100191,
PR China
c School of Public Health, Peking University Health Science Center, Beijing 100191, PR China
d Medical & Healthy Analysis Center, Peking University, Beijing 100191, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To maintain the virtue of good optical property and discard the dross of conventional fluorescent staining
dyes, we provide a strategy for designing new fluorescent scaffolds. In this study, a novel fluorescent
labeling glycoprotein (chitosan-poly-l-cysteine, CPC) was synthesized through graft copolymerization.
CPC gives emission peak at 465–470 nm when excited at 386 nm. The submicro-scale CPC microspheres
could be localized and persisted specifically in the cytoplasm of living cells, with strong blue fluorescence.
Moreover, CPC was highly resistant to photo bleaching, the fluorescence was remained stable for up to 72 h
as the cells grew and developed. The glycoprotein CPC was bio-compatible and in zero grade cytotoxicity
as quantified by MTT assay. The fluorescent labeling process with our newly designed glycoprotein CPC
is exceptionally efficient.
Received 25 October 2013
Accepted 28 August 2014
Available online 22 September 2014
Keywords:
Artificial fluorescent glycoprotein
Cytoplasmic labeling
Chitosan-poly-l-cysteine
Thiol group
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
of the first biological single cell lasers (Gather & Yun, 2011) and
the monitoring in time and space of various life phenomenon in
Fluorescent labeling technique has attracted much attention for
its wide application in biological and medical science. It serves as a
versatile and indispensable tool for flow cytometry, molecular and
cellular imaging, etc. (Boonacker & Van Noorden, 2001; Goncalves,
2009; Johnson, 1988; Kobayashi, Ogawa, Alford, Choyke, & Urano,
2010; Lavis & Raines, 2008; Petit, Denis-Gay, & Ratinaud, 1993;
Tinnefeld & Sauer, 2005; Walter, Huang, Manzo, & Sobhy, 2008;
Zhang, Campbell, Ting, & Tsien, 2002). The interests of structurally
diverse small-molecular fluorescent dyes and genetically encoded
fluorescent proteins such as “green fluorescent protein (GFP) fam-
ily” with different spectroscopic properties, realize the discovery
living cells and organisms like gene expression, protein localiza-
tion and dynamics, chromosome replication and cell division (Cha,
Snyder, Selvin, & Bezanilla, 1999; Chalfie, Tu, Euskirchen, Ward, &
Prasher, 1994; Giepmans, Adams, Ellisman, & Tsien, 2006; Goddard
& Reymond, 2004; Lampert & Westermann, 2011; Topf, Theis, &
Stockem, 1996; Willoughby & Cooper, 2008). So far, most of the
fluorescent dyes are used for staining nucleic acid, bilayer lipid
and cellular organelle. Few attentions have been paid to the spe-
cific cytoplasmic labeling. Cytoplasmic fluorescent labeling is an
effective and powerful tool for the exploration of cell membrane
integrity, long-term cell tracing, drug transport and distribution
(Brisson et al., 2011; Hawkins et al., 2010; Wang et al., 2011).
Whereas, at present, the specific cytoplasmic fluorescent labeling
compounds are quite few and are mainly limited to rigid poly-
cyclic aromatic dyes, such as Calcein-AM, BCECF-AM and CFSE,
and most of them are restricted in staining living cell for their
high toxicity. Thus, it is necessary for further development of
fluorescent material for cytoplasmic labeling with good biocom-
patibility.
Abbreviations: GFP, green fluorescent protein; CPC, chitosan-poly-l-cysteine;
DP, the average polymerization degree; S-benzyl-l-cysteine-NCA, S-benzyl-l-
cysteine-N-carboxy-anhydride; CHL cells, Chinese hamster lung cells; PBS,
phosphate-buffered saline; CLSM, confocal laser scanning microscopy; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
∗
Corresponding author at: Beijing Key Laboratory of Bio-Inspired Energy Materi-
als and Devices, School of Chemistry and Environment, Beihang University, Beijing
100191, PR China. Tel.: +86 10 82339539; fax: +86 10 82339539.
E-mail address: xiangy@buaa.edu.cn (Y. Xiang).
To maintain the virtue of good optical property and discard
the dross of conventional fluorescent staining dyes, we provide
http://dx.doi.org/10.1016/j.carbpol.2014.08.118
0144-8617/© 2014 Elsevier Ltd. All rights reserved.

212
J. Si et al. / Carbohydrate Polymers 117 (2015) 211–214
Scheme 1. Synthesis of fluorescent glycoprotein (chitosan-poly-l-cysteine, CPC) [m, n represent the number of deacetylated and acetylated units respectively which were
randomly distributed in the chitosan molecular chains. p represents the number of the poly-l-cysteine grafted on the chitosan].
here a strategy for designing a new fluorescent compound as
a specific cytoplasmic labeling material with low-toxicity, by
employing low molecular weight (MW) chitosan as a scaffold and
poly-l-cysteine as side chains, to form a well-defined fluorescent
glycoprotein (chitosan-poly-l-cysteine, CPC) (Xiang, Si, Zhang, Liu,
& Guo, 2009). Here, thiol groups on the side chains were used as
the important chromophores besides pyranose rings of chitosan
scaffold.
shift (85–90 nm), which is benefiting for signal detection and
imaging applications (shown in Supporting Information Fig. S2).
The submicro-scale glycoprotein CPC microspheres dispersing in
water showed strong blue fluorescent signal (Fig. S3).
CHL cells, with low phagocytic ability and reproduction rate,
were selected to study the fluorescent properties of CPC in the
living cells. During 48 h of co-incubation, the glycoprotein CPC
undergone the following three steps to be internalized in CHL
cells: (i) the aggregation of CPC microsphere around the living
CHL cells (Fig. S4); (ii) small microspheres entered the living
cells, exhibited by the appearance of a few bright fluorescent
spots in the living cells (Fig. 1a); (iii) a great number of micro-
spheres entered and aggregated in the living cells, resulting in
the appearance of stronger fluorescent bulks (Fig. 1b). The fluo-
rescent intensity of CHL cells increased correspondingly with the
time of co-incubation, as shown in Fig. 1c. Notably, after 48 h of co-
incubation, CHL cells regained their original peculiar spindle shape
from morbid round shape, indicating glycoprotein CPC was low-
toxic and the CHL cells could gradually adapt to the environment
with the addition of glycoprotein CPC. The cytotoxicity of glyco-
protein CPC was further quantified by MTT assay (Magrez et al.,
2006) with the result of zero grade (Fig. S6), much lower than
that of the traditional fluorescent dyes. On the other hand, the
CHL cells remained stably labeled for up to 72 h as they grew and
developed, indicating the glycoprotein CPC also have high photo-
stability.
2. Experimental
2.1. Synthesis of CPC
The whole synthesis process of CPC includes the following
four steps: the protection of thiol by benzyl group, S-benzyl-
l-cysteine-N-carboxy-anhydride (NCA), graft copolymerization
between chitosan and NCA and removing of the protective
group.
2.2. The culturing of Chinese hamster lung (CHL) cells
The detailed culturing process is shown in the supporting infor-
mation.
3. Results and discussion
Fig. 2 illustrates the localization of glycoprotein CPC micro-
spheres in the living CHL using confocal laser scanning microscopy
(CLSM). Results show the main regions of CHL cell were well
partitioned. The glycoprotein CPC, represented with strong blue flu-
orescence (Fig. 2a), was specifically localized in cytoplasmic region
of living CHL cells. On the other hand, the central area, labeled in
green (Fig. 2b) and red (Fig. 2c), represented nucleic zone stained
with Acridine Orange (AO), a nucleic acid-specific dye; the limited
red spots (Fig. 2d) were RNA in cytoplasm of CHL cells. The CLSM
The glycoprotein CPC was synthesized according to the route
as shown in Scheme 1. The average polymerization degree (DP)
of poly-l-cysteine side chains and the average MW of CPC were 7
and 46.66 kDa, respectively, which were adjustable by controlling
the different ratio between chitosan and S-benzyl-l-cysteine-N-
carboxy-anhydride (S-benzyl-l-cysteine-NCA).
In this study, glycoprotein CPC exhibited a unique photophys-
ical property. When excited at 380 nm, glycoprotein CPC gives
a maximum emission peak at 465–470 nm with a large Stokes

J. Si et al. / Carbohydrate Polymers 117 (2015) 211–214
213
Fig. 1. Fluorescent images of living CHL cells co-incubated with glycoprotein CPC microspheres for (a) 12 h, (b) 48 h and (c) the quantitative fluorescent intensity of living
CHL cells increased along with the increasing co-incubation time.
Acknowledgements
This research was financially supported by grants from
the National Basic Research Program of China (973 pro-
gram) (2011CB935700), National Natural Science Foundation of
China (U1137602, 51422301, 21373022), Beijing Novel Program
(2008B12). We thank T.T. Zhao at Beihang University for design-
ing of the cell culture and labeling experiment. We thank J. Zhang
at Beihang University and Y.S. Si at Agricultural University of Hebei
for the calculation of fluorescence intensity.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.carbpol.
2014.08.118.
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