1

Artifi cial Cells, Nanomedicine, and Biotechnology, 2013; Early Online: 1–6

Copyright © 2013 Informa Healthcare USA, Inc.

ISSN: 2169-1401 print / 2169-141X online

DOI: 10.3109/21691401.2013.841173

The liver-targeting study of the N-galactosylated chitosan in vivo and in vitro

Meihao Liang 1 , Xiaoliang Zheng 2 , Linglan Tu 2 , Zhen Ma 1 , Zunyuan Wang 1 , Dongmei Yan 2 & Zhengrong Shen 1

1 Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou, P. R. China, and 2 Center for Molecular

Medicine, Zhejiang Academy of Medical Sciences, Hangzhou, P. R. China

Introduction

Asialoglycoprotein receptor (ASGPR) is a transmembrane

protein, and consists of two diff erent subunits H1 and H2

(Ashell and Harford 1982). ASGPR mainly exists on the

mammalian polygonal cell surface, and specifi cally recog-

nizes the termini of galactose or N-acetylglucosamine resi-

dues of desialylated glycoprotein (Pricer and Ashwell 1971).

Th erefore, liver-targeting drug delivery may be achieved by

conjugating the carriers to a ligand that can bind to ASGPR

(Park et al. 2003, Rensen et al. 1997).

Chitosan (CS) is assembled with N-acetyl-glucosamine

and glucosamine residues by (1, 4)- β -glycosidic bonds.

Because of its biocompatibility, biodegradability as well as

non-toxicity properties etc, CS has been widely used as drug

carriers or gene vectors in the forms of tablets, capsules, pel-

lets, beads, microspheres, micro, and nano-particles etc (Sand-

ford 1989, Rao and Sharma 1997). A series of low molecular

weight (Mw)-galactosylated polymers have been reported

active or passive targeting the liver, in the way of being drug

carriers or gene vectors (Jeong et al. 2005, Park et al. 2000).

In our previous work, we reviewed the liver targeting

mediated by ASGPR (Liang et al. 2010), and synthesized

a series of N-galactosylated chitosan (GC) with diff erent

degree of substitution (DS) of lactosyl group, the macromo-

lecular pro-drug containing 5-fl uorouracil with synthesized

GC as carrier (Liang et al. 2011). In this study, we focused

on the liver-targeting specifi city of the GC derivatives con-

taining diff erent galactose moieties. GC with diff erent DS

of lactosyl groups was fi rst synthesized under a variety of

reaction conditions. Th e expression levels of ASGPR were

determined in vitro by western blot. Th e viability of cell

was also determined using 3-(4,5- Dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) assay. Finally the

distribution and localization of fl uorescein isothiocyanate

(FITC)-labeled GC (GC-FITC) in vivo were evaluated using

the fl uorescence measurement.

Materials

Th e Institute of Cancer Research (ICR) mice (male and

female, 18 – 22 g) were provided by experimental animal cen-

ter of Zhejiang Province (Hangzhou, Zhejiang, China). Th e

human normal liver cell line HL-7702 and hepatoma cell

lines SMMC-7721, HepG2 were obtained from Shanghai Cell

Bank, Chinese Academy of Sciences (Shanghai, China).

CS was obtained from Qingdao Honghai Biotechnology

Co., Ltd (Qingdao, Shandong, China) with 95% deacetyla-

tion degree and Mw 20 kda. Lactobionic acid (LA), FITC,

and 1-ethyl-3- (3-trimethyl- amine-propyl) carbodiimide

hydrochloride (EDC•HCl) were purchased from Aladdin ’ s

Correspondence: Zhengrong Shen, Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, P. R. China. E-mail: shenzr601@

163.com

(Received 9 July 2013 ; revised 20 August 2013 ; accepted 2 September 2013 )

Abstract

In order to study the liver targeting of the N-galactosylated

chitosan (GC) polymer in liver, we fi rst conjugated the

lactobionic acid with chitosan (CS) to obtain the carrier of GC

with diff erent degree of substitution of lactosyl group. Western

blot was performed to detect the expression levels of the

asialoglycoprotein receptors (ASGPR) in the cell lines of HepG2,

SMMC-7721, and HL-7702. The protein level of ASGPR was lower

in HepG2 compared to HL-7702 and SMMC-7721. Although

all treated by CS, viabilities of HL-7702 and HepG2 did not

experience any signifi cant drop, while viability of SMMC-7721

decreased 15% on average from control. It was the fi rst data

about the inhibitory eff ect of GC on the liver cells. Fluorescein

isothiocyanate (FITC) labeled GC (GC-FITC) was injected

intravenously into mice at a dose of 0.02 μ mol/mouse. GC-FITC

showed maximum liver localization at 5 min and even detectable

at 48 h after injection. Further, the accumulation of GC in liver

was about 5.4-fold higher than that of CS. In conclusion, GC

demonstrated its higher effi cacy in drug liver targeting and

thus could be a more promising drug or gene carrier in future

therapies.

Keywords: asialoglycoprotein , receptor , chitosan , liver-targeting ,

N-galactosylated chitosan , polymer carrier

Art

ific

ial C

ells

, Nan

omed

icin

e, a

nd B

iote

chno

logy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nyu

Med

ical

Cen

ter

on 1

1/29

/13

For

pers

onal

use

onl

y.

2 M. Liang et al.

Corporate (Shanghai, China). Dulbecco ’ s modifi ed eagle

medium (DMEM) and trypsin were purchased from Gibco

(Grand Island, NY, USA). Fetal bovine serum was commer-

cially obtained from Zhejiang Tianhang biological technol-

ogy Co. Ltd. (Hangzhou, Zhejiang, China). MTT and dextran

were obtained from Sigma-Aldrich (St. Louis, MO, USA).

ASGPR1/2 (FL-291) was purchased from Santa Cruz Bio-

technology (Danvers, MA, USA). Beta-Actin ( β -Actin) and

glyceraldehyde-3- phosphate dehydrogenase were obtained

from Cell Signaling technology Co. Ltd. (Danvers, MA,

USA). All other chemicals were analytical grade and pur-

chased from Hangzhou Huipu Chemical Instrument Co. Ltd.

(Hangzhou, Zhejiang, China).

Methods

Synthesis of GC GC was synthesized based on the method in the literature

(Liang et al. 2010). LA (1.1 – 2.2 g) and EDC • HCl (1.4 – 2.8 g)

were added to a mixture of CS (1.0 g) hydrochloric acid solu-

tion 0.5% (v/v) (40 mL). Th e mixture was stirred for 48 – 72 h

at 30 ° C. Th e resulting product was purifi ed using a dialysis

tube against distilled water for 72 h, followed by lyophiliza-

tion for 44 h. According to the catalyst feeding amount and

reaction time diff erences, three samples: high level DS

of lactosyl group in GC (GCH), middle level DS of lactosyl

group in GC (GCM), and low level DS of lactosyl group in

GC (GCL), were prepared, respectively. Th eir structure was

confi rmed by proton nuclear magnetic resonance ( 1 H-NMR)

spectra using a Bruker Avance 400 spectrometer. GCH, 1 H-

NMR (D 2 O) δ : 1.87 (3H, s), 3.38 ~ 3.77 (16H, m), 4.06 (1H, s),

and 4.38 (1H, m). 1 H-NMR spectra of GCM and GCL were

similar to the spectra of GCH. Th e synthetic scheme of GC

was made using ChemDraw Ultra 7.0.

Th e Mw of the polymer was determined using gel perme-

ation chromatography (GPC).GPC was performed at room

temperature using a high-performance liquid chromatogra-

phy pump equipped with diff erential refractive index detec-

tor (models 515 and 2410, respectively, both from Waters,

Milford, MA, USA). Tsk-Gel G4000SWXL (7.8 � 300 mm)

(Tosoh, Tohoku, Japan) was used as a column. Th e buff er

solution containing 0.1 M sodium acetate and 0.1 M acetic

acid, pH � 4.8, was used as the elution solvent at a fl ow of

0.8 mL/min. Dextran was used as a Mw calibration marker,

and its peak Mws (MP) are 2.0 � 10 6 , 1.88 � 10 5 , 7.69 � 10 4 ,

1.05 � 10 4 , and 3.2 � 10 3 . Th e values of DS of lactosyl

group in GC and the Mw of GC are shown in Table I using

Microsoft Word.

Conjugation of GCH and CS with FITC GCH (0.5 g) was dissolved in 0.1M sodium bicarbonate buff er

(50 ml, pH 9.0). Th irty-fi ve milligram of FITC was dissolved

in 50 ml acetone, and then slowly added to the solution

above. Th e mixture was stirred at 40 ° C for 18 h in dark and

was then puri fi ed using a dialysis tube against distilled water

until no fl uorescence was detected in the dialysate, followed

by lyophilization, obtaining GCH labeled with FITC (GCH-

FITC). Th e labeling effi ciency of GCH-FITC was 1.34% (w/w),

determined by spectrophotometry at 490 nm wavelength.

CS labeled with FITC (CS-FITC) was synthesized based on

the method described above, and the labeling effi ciency of

CS-FITC was 1.42% (w/w).

Body distribution and biodegradability of GCH-FITC in mice After intravenous administration (i.v.) of GCH-FITC or CS-

FITC in ICR mice at a dose of 0.2 mL (0.1 mM), the mice

were sacrifi ced at 5 min, 30 min, 1 h, 8 h, 24 h, and 48 h time

points, and the heart, liver, spleen, lung, and kidney were

collected and accurately weighed. A 10-fold volume of

phosphate-buff ered saline (PBS) was added, and the mix-

ture was homogenized on ice with a glass homogenizer. Th e

fl uorescence intensities in tissue homogenates were deter-

mined by multifunctional micro plate detection (Ex � 489

nm, Em � 520 nm). Th e distribution levels of GCH or CS

were calculated using the standard calibration curve. Tis-

sue distribution of GCH-FITC and CS-FITC in ICR mice is

presented in a graph in Figure 2 using Microsoft Excel.

To preparing the standard calibration curve, the liver,

spleen, lung, and kidney of normal ICR mice was, respec-

tively, homogenized and mixed with PBS and GCH-FITC

Table I. Th e values of DS of lactosyl group in GC and the Mw of GC.

Sample DS(%) Mw(kDa)

GCH 37.0 47GCM 24.2 35GCL 12.9 23CS / 20

Figure 1. Th e synthetic scheme of GC (Liang et al., 2011).

Art

ific

ial C

ells

, Nan

omed

icin

e, a

nd B

iote

chno

logy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nyu

Med

ical

Cen

ter

on 1

1/29

/13

For

pers

onal

use

onl

y.

The Liver-targeting of GC 3

trimethyl ammonium bromide, 40 mM NaCl, 0.6 mM

ethylene diamine tetraacetic acid, 3 mM Tris-HCl, and

0.02 mM phenylmethanesulfonyl fluoride (PMSF), and

centrifuged (12,000 rpm) for 10 min at 4 ° C. After protein

quantification, the extracted protein was subjected to

sodium dodecyl sulfate polyacrylamide gel electropho-

resis (SDS-PAGE) in a 10% (v/v) sodium dodecyl sulfate

(SDS) gel, and the protein was transferred to nitrocellu-

lose membrane. After blocking, the blots were incubated

for 1 h at 25 ° C separately with the primary antibodies of

ASGPR1/2 (FL-291) at 1:200 for 2 h, followed by incuba-

tion with secondary antibody (goat anti-rabbit IgG-HRP)

at a 1:1000 dilution for 2 h. Immunoreactive bands of

ASGPR1/2 (FL-291) and β -actin mark were visualized

using enhanced chemiluminescence reagents. ASGPR1/2

(FL-291) and β -actin mark activities were quantified by

densitometry of autorad- iographs (Bio-rad, CA, USA).

ASGPR expression in the human normal liver cell line is

shown graphically in Figure 4 using Microsoft Excel.

Cell viability assay Th e cell lines of HL-7702, SMMC-7721, or HepG2 were

plated out at a density of 7.5 � 10 3 per well in 96-well fl at-

bottom plates and allowed to attach and grow for 24 h. CS,

GCH, GCM, or GCL was added at the required concentra-

tion (10 � 6 M, 10 � 7 M, and 10 � 8 M). After a 72-h incuba-

tion in continuous drug exposure, MTT (100 μ L, 1 g/L) was

added to each well. Plates were kept in the dark at 37 ° C for

4 h. After removing the medium and MTT, MTT-formazan

crystals were dissolved in acidifi ed isopropanol contain-

ing 0.04 mol/L HCl (150 μ L/well). Th e plates were kept in

the dark for 30 min, and the absorbance was measured at

570 nm with multi-well plate reader (Model 680, Bio-Rad).

Th e proliferation inhibition of CS, GCH, GCM, and GCL on

the cell lines is shown graphically using Microsoft Excel. Th e

percentage of cell viability was calculated according to the

following formula:

Percentage of cell viability � (absorbance value at tested

well/absorbance value at control well) � 100%.

Statistical methods and strategies Data are expressed as mean � SD. Statistical signifi cance of

the diff erences between groups was analyzed by one-way

Figure 2. Tissue distribution of GCH-FITC and CS-FITC in ICR mice at 5 min, 1 h, and 8 h after i.v. administration at a dose of 0.2 mL per mouse. Each point represents the mean � SD ( n � 3).

or CS-FITC separately to obtain 1, 10, and 100 μ M stan-

dard solution. The fluorescence intensity of the standard

solution was measured with the multifunctional enzyme

immunoassay (Ex � 489 nm, Em � 520 nm).

Expression of ASGPR in human normal liver and hepatoma cell lines The cells of HL-7702, SMMC-7721, and HepG2 were

cultured at 37 ° C in DMEM supplement containing 10%

(v/v) fetal calf serum, 100 U/L penicillin, and 100 mg/L

streptomycin in 5% (v/v) CO 2 . For total cellular protein,

cells were lysed in buffer containing 1.6 mM hexadecyl

Figure 3. Th e biodegradability of CS-FITC and GCH-FITC in liver of ICR mice after i.v. at a dose of 0.2 mL per mouse. Each point represents the mean � SD ( n � 3).

Art

ific

ial C

ells

, Nan

omed

icin

e, a

nd B

iote

chno

logy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nyu

Med

ical

Cen

ter

on 1

1/29

/13

For

pers

onal

use

onl

y.

4 M. Liang et al.

ANOVA followed by Newman – Keuls multiple comparisons

tests using SPSS 10.0 for windows. * P � 0.05 (or Δ P � 0.05)

was considered statistically signifi cant.

Results and discussion

Synthesis of GC GC was synthesized as previously reported (Liang et al.

2011). Figure 1 shows the synthetic approach. Th e SD of

lactosyl group in GC was calculated by comparing the

characteristic peak areas of LA group with original acet-

amide group of CS. By changing the feed ratio of LA and

the reaction time, the diff erent DS of lactosyl group in GC

was obtained, namely GCH, GCM, and GCL. Th e values of

lactosyl group in GC and the Mw of GC and CS are listed

in Table I.

Body distribution of GCH-FITC in mice Figure 2 shows the percentage of GCH-FITC and CS-FITC

in liver, spleen, lung, and kidney at 5 min, 1 h, and 8 h after

i.v. at a dose of 0.2 mL per mouse. Th e amount of GCH-FITC

Figure 5. Th e proliferation inhibition of CS, GCH, GCM, and GCL on the cells of SMMC-7721 (A), HepG2 (B) determined by MTT assay. (mean � SD, n � 8). * P � 0.05, * * P � 0.01 versus control; D P � 0.05,versus CS.

Figure 4. ASGPR expression in the human normal liver cell line HL-7702 and hepatoma cell line HepG2 or SMMC-7721 by Western blotting (A). Quantitative analysis of ASGPR expression in diff erent cell lines (B) (mean � SD, n � 3).

Art

ific

ial C

ells

, Nan

omed

icin

e, a

nd B

iote

chno

logy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nyu

Med

ical

Cen

ter

on 1

1/29

/13

For

pers

onal

use

onl

y.

The Liver-targeting of GC 5

in liver was 28.4% (w/w), 21.9% (w/w), and 22.2% (w/w)

at 5 min, 1 h, and 8 h respectively, while 5.2% (w/w), 3.3%

(w/w), and 2.9% (w/w) for CS-FITC. Notably, the percentage

of GCH-FITC or CS-FITC in spleen and lung was very low.

In kidney, GCH-FITC was gradually reduced from 43.0%

to 26.2% of the dose from 5 min to 8 h, while from 66.0%

(w/w) to 46.6% (w/w) for CS-FITC. Th us, from the data in

Figure 2 it can be deduced that GCH-FITC is not combined

with other organs, and will be mainly excreted through the

kidney. Th e same results also proved that GCH group by our

preparation could maintain high accumulation in mice ’ s

liver tissue.

Biodegradability of GCH-FITC in liver Th e body distribution of GCH-FITC and CS-FITC was deter-

mined in liver tissue after the treatment (intravenously) of

the compounds dissolved in saline (1 � 10 � 4 M) at a dose

of 0.2 mL per mouse (Figure 3). Th e concentrations of

GCH-FITC and CS-FITC reached the maximum amount at

5 min and fell down slowly, especially at 30 min ( * P � 0.05).

Interestingly, the peak concentration of GCH-FITC in liver

was 5.4 times more than that of the CS-FITC. After 30 min,

however, there was no diff erence for GCH-FITC in liver tis-

sue ( * P � 0.05). It was proved that the lactose residues in GC

could promote the aggregation of GC in the liver tissue and

less biodegradation.

ASGPR expression in human normal liver and hepatoma cell line by western blot Th e ASGPR expression in human normal liver and hepa-

toma cell line is shown in Figure 4. Th e expression levels of

ASGPR in SMMC-7721 and HepG2 were 115.8% and 18.9%,

respectively normalized to HL-7702 as the standard (100%).

Th e results demonstrated that SMMC-7721 expressed more

abundant ASGPR than HepG2.

Uptake of GC and CS by hepatoma cell Th e experiment was divided into fi ve groups: GCH group,

GCM group, GCL group, CS group as the negative control,

and the culture solution as blank. Th e experiment results are

shown in Figure 5. Th e concentrations of GCH, GCM, GCL,

and CS were 10 � 8 M, 10 � 7 M, and 10 � 6 M, respectively.

Th e CS is known for demonstrating antitumor eff ect on

hepatoma cells (Suzuki et al. 1986). Th us the extent of CS

inhibitory eff ect to hepatoma cell viability refl ects its hepa-

toma cellular uptake rate. As indicated in Figure 5A, the

inhibitory eff ect of GCH is stronger compared with that of

CS, and all treated by CS or GCH at the concentration of 10 � 6

M, viabilities of HL-7702 and HepG2 did not experience any

signifi cant drop, while viability of SMMC-7721 decreased

signifi cantly. However, as shown in Figure 5B this was not

the case for HepG2 cells. Such a phenomenon is intrigu-

ing and is possibly because SMMC-7721 has demonstrated

more abundant ASGPR expression than HepG2 (Figure 4).

Th is higher level of ASGPR expression may lead to higher

GC uptake rate, and thus lead to more resultant inhibition to

SMMC-7721 than to HepG2. Together with eff ects of the DS

of lactose residues, all these results suggested that the inhibi-

tory eff ects of CS to hepatoma cell are tightly infl uenced by

LA conjugation and ASGPR expression.

Toxicity of GC and CS in human normal liver cell line HL-7702 From the cytotoxicity MTT assay results in Figure 6, it is

evident that the viability of HL-7702, an ASGPR-expression

human liver cell line, was not aff ected by GC and CS. Th is

suggested that CS and GC presented non-toxicity to normal

liver cells.

Conclusions

GC specifi cally accumulated in the liver with a long-term sta-

bility and was uptaken by hepatoma cell through the ASGPR,

and has no toxicity in normal liver cells. To our knowledge,

this was the fi rst data report of GC ’ s inhibitory eff ects on both

normal liver and hepatoma cell lines. Th ese results therefore

provide valuable information to develop the chitosan drug

conjugate systems in the future.

Declaration of interest

Th e authors report no declarations of interest. Th e authors

alone are responsible for the content and writing of the

paper.

Th is work was supported by Natural Science Foundation

of Zhejiang Province (Grant No.Y207790), Health Bureau of

Zhejiang Province (Grant No.XKQ-01001), and Science and

Technology Department of Zhejiang Province (Grant No.

2012F10005).

References

Ashell G , Harford J . 1982 . Carbohydrate-specifi c receptors of the liver . Annu Reu Biochem. 51 : 531 – 554 .

Jeong YI , Seo SJ , Park IK , Lee HC , Kang IC , Akaike T , Cho CS . 2005 . Cellular recognition of paclitaxel-loaded polymeric nanoparticles composed of poly(gamma-benzyl-l-glutamate) and poly(ethylene glycol) diblock copolymer endcapped with galactose moiety . Int J Pharm. 296 : 151 – 161 .

Liang MH , Ma Z , Wang ZY , Shen ZR . 2011 . Synthesis and characteriza-tion of n-galactosylated-chitosan-5-fl uorouracil acetic acid conju-gate . Chin Pharm J. 46 : 1677 – 1680 .

Figure 6. Th e proliferation inhibition of CS, GCH, GCM, and GCL on the normal liver cell line HL-7702 determined by MTT assay. (mean � SD, n � 8).

Art

ific

ial C

ells

, Nan

omed

icin

e, a

nd B

iote

chno

logy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nyu

Med

ical

Cen

ter

on 1

1/29

/13

For

pers

onal

use

onl

y.

6 M. Liang et al.

Rensen PC , Herijgers N , Netscher MH , Meskers SC , Eck M , Berkel TJ . 1997 . Particle size determines the specifi city of apo-lipoprotein e-containing triglyceride-rich emulsions for the ldl receptors versus hepatic remnant receptor in vivo . J Lipid Res. 38 : 1070 – 1084 .

Sandford P . 1989 . Chitosan: commercial uses and potential applications . Skj å kbraek G, Anthonsen T, Sandford P, Eds . Chitin and Chitosan. London, UK: Elsevier Applied Science , pp. 51 – 69 .

Suzuki K , Tokoro A , Okawa Y , Suzuki S , Suzuki M . 1986 . Eff ect of N-acetylchito-oligosaccharides on activation of phagocytes . Microbiol Immunol. 30 : 777 – 787 .

Liang MH , Shen ZR , Ma Z , Wang ZY . 2010 . Progress on ASGPR-mediated hepatic targeting . Chin J Mod Appl Pharm. 2 : 109 – 114 .

Park IK , Yang J , Jeong HJ , Bom HS , Harada I , Akaike T , et al . 2003 . Galactosylated chitosan as a synthetic extracellular matrix for hepa-tocytes attachment . Biomaterials. 24 : 2331 – 2337 .

Park YK , Park YH , Shin BA , Choi ES , Park YR , Akaike T , Cho CS . 2000 . Galactosylated chitosan-graft-dextran as hepatocyte-targeting DNA carrier . J Controlled Release. 69 : 97 – 108 .

Pricer WE , Ashwell G . 1971 . Th e binding of desialylated glycoproteins by plasma membranes of liver . J Biol Chem. 246 : 4825 – 4833 .

Rao SB , Sharma CP . 1997 . Use of chitosan as a biomaterial: studies on its safety and hemostatic potential . J Biomed Mater Res. 34 : 21 – 28 .

Art

ific

ial C

ells

, Nan

omed

icin

e, a

nd B

iote

chno

logy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Nyu

Med

ical

Cen

ter

on 1

1/29

/13

For

pers

onal

use

onl

y.