lectin-mediated cytotoxicity and specificity of 5-fluorouracil conjugated with peanut agglutinin...

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Lectin-mediated cytotoxicity and specificity of 5-fluorouracil conjugated with peanut agglutinin (5-Fu-PNA) in vitro QIN CAI, & ZHI-RONG ZHANG Key Laboratory of Drug Targeting and Novel Drug Delivery Systems, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P.R. China (Received 30 November 2004; revised 21 January 2005; accepted 30 March 2005) Abstract In order to take advantage of the biorecognition between lectin and carbohydrate for targeted drug delivery, the lectin of peanut (Arachis hypogaea) agglutinin (PNA) was coupled by fixing its amino groups to the carbodiimide-activated carboxylic groups of 5-fluorouracil (5-Fu) derivative (N 1 -substituted 5-Fu acetate) to form 5-Fu-PNA conjugate. When the coupling reaction was carried out in the presence of D-galactose (D-gal, specific sugar for PNA), the affinity of PNA was maintained after its coupling to N 1 -substituted 5-Fu acetate, which was confirmed by the result of the haemagglutination test. Otherwise, PNA would lose its affinity after the cross-linking reaction. The cytotoxicity, specificity and selectivity of 5-Fu-PNA were examined on the human colorectal cancer cell line LoVo and the human normal liver cell line Chang using MTT assay. Compared with free drug, the active conjugate, which maintained the affinity of lectin, had similar cytotoxic effect on LoVo cells with much lower cytotoxicity on Chang cells ðp , 0:05Þ: On the other hand, lower cytotoxic effects on LoVo cells were observed for the non-active conjugate even at higher drug concentrations. The cytotoxic effect of conjugate was specific because only the active conjugate could inhibit the growth of LoVo cells in a dose- and time-dependent manner as that of the free drug. The achieved results indicate the significance to maintain the affinity of lectin for lectin-mediated cytotoxicity. Still, the potential of 5-Fu-PNA conjugate as a targeting agent for colorectal cancer needs to be further investigated in vivo. Keywords: Lectin, 5-Fu-PNA conjugate, targeted drug delivery, cytotoxicity Introduction Cancer chemotherapy often causes unwanted side effects due to the low selectivity of chemotherapeutic agents for cancer cells. Drug targeting, which could carry drugs directly to the target sites via specific ligands against receptors expressed on malignant cells, is one approach to overcome this problem. However, reali- zation of targeted drug delivery was hampered by the difficulties in finding appropriate carrier molecules. An appropriate carrier molecule plays an important role for a successful targeted delivery system. Cancerous cells often express different glycans, compared with their normal counterparts (Shanghal and Hakamori 1990). Lectin recognizes glycans on cell surface with a high degree of specificity. Therefore, lectins are proposed to be promising carrier molecules to target drugs specifically to different cells and tissues (Bru ¨ck et al. 2001, Yi et al. 2001, Wirth et al. 2002). In literatures, lectin-mediated drug delivery systems have showed their advantages (Russell-Jones et al. 1999, Smart 2004, Jepson et al. 2004). The lectin of peanut (Arachis hypogaea) agglutinin (PNA) is a tetramer, carbohydrate-free protein composed of four identical subunits exhibiting a molecular weight of 110 kDa (Lotan et al. 1975). PNA has been extensively used as a probe to detect malignant phenotype in several tissues as it strongly recognizes the cancer specific T antigen ISSN 1061-186X print/ISSN 1029-2330 online q 2005 Taylor & Francis Group Ltd DOI: 10.1080/10611860500138505 Correspondence: Z. -R. Zhang, Key Laboratory of Drug Targeting and Novel Drug Delivery Systems, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P.R. China. Tel: 86 28 85501566. Fax: 86 28 85456898. E-mail: [email protected] Journal of Drug Targeting, May 2005; 13(4): 251–257 Journal of Drug Targeting Downloaded from informahealthcare.com by Michigan University on 11/03/14 For personal use only.

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Page 1: Lectin-mediated cytotoxicity and specificity of 5-fluorouracil conjugated with peanut agglutinin (5-Fu-PNA)               in vitro

Lectin-mediated cytotoxicity and specificity of 5-fluorouracil conjugatedwith peanut agglutinin (5-Fu-PNA) in vitro

QIN CAI, & ZHI-RONG ZHANG

Key Laboratory of Drug Targeting and Novel Drug Delivery Systems, West China School of Pharmacy, Sichuan University,

No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P.R. China

(Received 30 November 2004; revised 21 January 2005; accepted 30 March 2005)

AbstractIn order to take advantage of the biorecognition between lectin and carbohydrate for targeted drug delivery, the lectin ofpeanut (Arachis hypogaea) agglutinin (PNA) was coupled by fixing its amino groups to the carbodiimide-activated carboxylicgroups of 5-fluorouracil (5-Fu) derivative (N1-substituted 5-Fu acetate) to form 5-Fu-PNA conjugate. When the couplingreaction was carried out in the presence of D-galactose (D-gal, specific sugar for PNA), the affinity of PNA was maintainedafter its coupling to N1-substituted 5-Fu acetate, which was confirmed by the result of the haemagglutination test. Otherwise,PNA would lose its affinity after the cross-linking reaction. The cytotoxicity, specificity and selectivity of 5-Fu-PNA wereexamined on the human colorectal cancer cell line LoVo and the human normal liver cell line Chang using MTT assay.Compared with free drug, the active conjugate, which maintained the affinity of lectin, had similar cytotoxic effect on LoVocells with much lower cytotoxicity on Chang cells ðp , 0:05Þ: On the other hand, lower cytotoxic effects on LoVo cells wereobserved for the non-active conjugate even at higher drug concentrations. The cytotoxic effect of conjugate was specificbecause only the active conjugate could inhibit the growth of LoVo cells in a dose- and time-dependent manner as that of thefree drug. The achieved results indicate the significance to maintain the affinity of lectin for lectin-mediated cytotoxicity. Still,the potential of 5-Fu-PNA conjugate as a targeting agent for colorectal cancer needs to be further investigated in vivo.

Keywords: Lectin, 5-Fu-PNA conjugate, targeted drug delivery, cytotoxicity

Introduction

Cancer chemotherapy often causes unwanted side

effects due to the low selectivity of chemotherapeutic

agents for cancer cells. Drug targeting, which could

carry drugs directly to the target sites via specific ligands

against receptors expressed on malignant cells, is one

approach to overcome this problem. However, reali-

zation of targeted drug delivery was hampered by the

difficulties in finding appropriate carrier molecules. An

appropriate carrier molecule plays an important role for

a successful targeted delivery system.

Cancerous cells often express different glycans,

compared with their normal counterparts (Shanghal

and Hakamori 1990). Lectin recognizes glycans on cell

surface with a high degree of specificity. Therefore,

lectins are proposed tobe promising carrier molecules to

target drugs specifically to different cells and tissues

(Bruck et al. 2001, Yi et al. 2001, Wirth et al. 2002).

In literatures, lectin-mediated drug delivery systems

have showed their advantages (Russell-Jones et al. 1999,

Smart 2004, Jepson et al. 2004).

The lectin of peanut (Arachis hypogaea) agglutinin

(PNA) is a tetramer, carbohydrate-free protein

composed of four identical subunits exhibiting

a molecular weight of 110 kDa (Lotan et al.

1975). PNA has been extensively used as a probe to

detect malignant phenotype in several tissues as

it strongly recognizes the cancer specific T antigen

ISSN 1061-186X print/ISSN 1029-2330 online q 2005 Taylor & Francis Group Ltd

DOI: 10.1080/10611860500138505

Correspondence: Z. -R. Zhang, Key Laboratory of Drug Targeting and Novel Drug Delivery Systems, West China School of Pharmacy,Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P.R. China. Tel: 86 28 85501566. Fax: 86 28 85456898.E-mail: [email protected]

Journal of Drug Targeting, May 2005; 13(4): 251–257

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Page 2: Lectin-mediated cytotoxicity and specificity of 5-fluorouracil conjugated with peanut agglutinin (5-Fu-PNA)               in vitro

(Gal-1-3GalNAc-) (Hwang et al. 1999, Chacko and

Appukuttan 2001). In addition, PNA receptors were

highly expressed in gastric and intestinal (GI) cancer,

but was not expressed or weakly expressed on normal

tissues (Kuang and Lei 1993, Lu and Xu 1997). These

data suggest the excellent selectivity of PNA towards

GI cancer.

To exploit the lectin-sugar binding selectivity, PNA

was chosen as the carrier molecule for the targeting

delivery of cytostatic agents to the colorectal cancer

tissue. The 5-Fu was used as the model drug to obtain

a targeting conjugate. The conjugate was characteri-

zed with SDS-PAGE. The affinity of the conjugate

was tested using haemagglutination assay and the

drug/lectin molar ratio was determined with TNBS

method. In order to examine the effectiveness of the

targeting system in comparison to the free drug, the

cytotoxicity of the drugs were assayed on LoVo cells

using MTT assay. The cytotoxic selectivity of the free

drug and the active conjugate was detected on normal

Chang cells. The specificity of the active conjugate was

also examined using MTT assay.

Materials and methods

Chemicals and animals

N-(3-dimethylaminopropyl)-N0-ethyl carbodiimide

hydrochloride (EDC), N-hydroxy succinimide (NHS),

N-morpholino propanesulfonic acid (Mops), trinitro-

benzenesulfonic acid (TNBS) and 3-(4,5-dimethyl-2-

tetrazolyl)-2,5-diphenyl-2H tetrazolium bromide

(MTT), were purchased from Sigma (St. Louis, MO,

USA). The PNAwasobtained from VectorLaboratories

Inc. (Burlingame, CA, USA). Cell culture medium

RPMI-1640 and DMEM were from Gibco Co. (USA).

The 5-Fu was from Nantong pharmaceutical Co. Ltd.

(Jiangsu, P.R.China). D-galactose (D-gal) was supplied

by Amresco (USA). All other chemicals were of

analytical grade obtained commercially.

MiceC57BL/6were culturedbyExperimentalAnimal

Center of Huaxi, Sichuan University, P.R. China.

Conjugation of 5-Fu to PNA

N1-substituted 5-Fu acetate was synthesized in two

steps according to the literature (Dong and Yu 1996)

with modifications. First, 5-Fu (3.90 g), K2CO3

(2.76 g) and KI (0.5 g) were dissolved in 10 ml

of DMSO. After heated to 708C, the mixture

was treated with ethyl chloro acetate (3.68 g)

and stirred overnight. After evaporation of the

solvent, the residue was extracted with ethyl

acetate and purified by silica gel column chromato-

graph (ethyl acetate/cyclohexane ¼ 2:1) to obtain an

ethyl N1-substituted 5-Fu acetate (2.71 g, mp

158–1608C, 1H-NMR(CDCl3) d 8.77(1H,br,N3-

H), 7.20(1H,d,JH–F ¼ 5:25;C6-H), 4.27(2H,s,N1-

CH2), 4.26(2H,q,O-CH2)). Secondly, the ethyl

N1-substituted 5-Fu acetate were dissolved in 10 ml

of HCl and refluxed for 5 h. After the solvent was

evaporated off, the residue was recrystallized from

H2O.

Cross-linking of N1-substituted 5-Fu acetate to

PNA was carried out via EDC/NHS using a single-

step method as follows: N1-substituted 5-Fu acetate

was activated by reacting with EDC and NHS in a

buffered solution (0.05 M Mops, pH ¼ 5:5) for 3 h at

the room temperature to form an active ester

(Grabarek and Gergely 1990). The molar ratio of

the carboxylic acid groups, EDC and NHS was 1:5:2.

PNA was incubated with or without D-gal in Mops

(0.05 M, pH ¼ 7:5) solution for 0.5 h before its

addition to the active ester solution. Then the pH

was adjusted to 7.5 by stepwise addition of 1 M NaOH

solution. The cross-linking of active ester to PNA

lasted for 12 h at 48C. The reaction solution was

applied to a column of Sephadex G-25. The column

was eluted with a solution of PBS (0.01 M, pH 7.2) at

a rate of 5 ml/min. Gel filtration on Sephadex G-25

provided two peaks and the fraction of the first peak

was collected, tested and lyophilized. The property of

the conjugate was detected on SDS-PAGE, which was

performed at pH 8.9 in 15% acrylamide running gels

and 4% stacking gels in the presence of 0.1% sodium

dodecyl sulfate at 140 V for 4 h. The gels were stained

with Coomassie brilliant blue R250. The result was

analysed with the JEL-PRO software (Mediacyber-

netics, USA). The drug/lectin molar ratio was

determined by TNBS method (Morcol et al. 1997).

Haemagglutination test

The affinity of PNA after covalent coupling was

determined by haemagglutination test because the

lectin could agglutinate thymus cells from mice

C57BL/6 (Gu and Li 1983).

According to the requirements of the National Act

on the use of experimental animals (P.R. China), the

Sichuan University animal ethical experimentation

committee approved all procedure of the studies

in vitro.

Briefly, the thymus was excised after sacrificing and

rinsed in isotonic PBS (pH7.2). The thymus was

chipped into single cells and the cell concentration was

adjusted to 2% (w/v) with the same PBS ready to be

used. A measure of 25ml of the conjugate solutions for

each well was added to a “V-bottom” 96-well plate

and diluted successively. Thereafter, 25ml of the cell

suspension was added when shaking and the plate was

incubated for 1.5–2 h. Affinity was confirmed by

comparing the value of titre of dilution giving the last

visible agglutination against a positive control (PNA

solution) and negative control (25ml of the cell

suspension added with 25ml of PBS). Agglutination is

judged by the flocculation of cells, which appears as a

diffuse milk-white color. In the case of a negative

Q. Cai & Z.-R. Zhang252

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Page 3: Lectin-mediated cytotoxicity and specificity of 5-fluorouracil conjugated with peanut agglutinin (5-Fu-PNA)               in vitro

result, there is a bright milk-white spot in the bottom

of the well due to the sinking of non-agglutinated cells.

All tests were done in triplicate at 258C.

Cell culture

The human colon carcinoma cell line LoVo and

the human normal liver cell line Chang were obtained

from Shanghai Cell Institute, China Academy of

Sciences. LoVo cells and Chang cells were cultivated

in RPMI 1640 and DMEM, respectively with 10%

(v/v) fetal calf serum (FCS), penicillin (100 U ml21)

and streptomycin (100 U ml21). The cells were

cultured at 378C in a humidified 5% CO2/95% air

incubator. After growing to a confluent monolayer,

cells were passaged by removing the adherent cells

with trypsin (in buffered saline, pH 7.2). Cell viability

was assessed by means of the trypan blue exclusion

method.

In vitro cytotoxic effect of 5-Fu-PNA conjugate

MTT assay was employed to determine the number

of surviving cells. The cytotoxicity of 5-FU in vitro

was evaluated either as the free drug or the conjugated

form. MTT assay is based on the ability of the

mitochondrial enzymes in living cells to convert a

yellow MTT tetrazolium salt into a blue MTT

formazan thus the amount of formazan present is

proportional to the number of viable cells. MTT

assay detects living cells with a high degree of precision

(Gieni et al. 1995).

The experiment was processed as follows: Malig-

nant cells were continuously treated at various

concentrations. A single cell suspension was obtained

and the cell number (counted with a haemocytometer)

was adjusted to 4 £ 104 cells per ml with culture

medium containing 10% FCS. A measure of 100ml of

cell suspension were added into the wells of flat-

bottom 96-well plates. After the cells adhered, 10ml of

solution was added per well containing appropriate

amount of 5-FU either in free state or conjugated

form. The plates were incubated for periods of time

ranging from 24 to 120 h at 378C and the surviving

cells were assayed for reduction of MTT. Each sample

was assayed in triplicate at each time period. At

intervals of each experimental time, 20ml of MTT

solution (5 mg/ml) was added to each well and the

plate was placed in the incubator for 4 h. The culture

medium was removed by aspiration. Thereafter,

100ml of DMSO was added to each well to dissolve

the formazan crystals while slightly stirring the cells

using an automated shaker. The absorbance of the

suspension was measured at 570/630 nm on an ELISA

plate reader (Bio-Rad, Microplate Reader 550). All

the results were expressed as % cell survival of the

control (untreated) over time. Background absor-

bance levels, determined in wells containing no cells,

were subtracted from the experimental and control

values.

Specificity of 5-Fu-PNA conjugate

In order to confirm the specificity of PNA-mediated

cytotoxic effect of 5-Fu, PNA, the active conjugate or

the non-active conjugate was added to LoVo cells,

respectively. The active and non-active conjugate were

obtained with or without the D-gal protection when

prepared, and the drug/lectin molar ratio of the non-

active conjugate was higher than that of the active

conjugate (see Results section). In brief, the cells were

seeded into 96-well plate at a concentration of

4 £ 103 cells per well. A measure of 10ml of solution

containing PNA, the active conjugate or the non-

active conjugate at various concentrations was added,

respectively, into the adherent cells with equivalent

amounts of lectin for 48 h or at certain concentration

for different incubation times. Subsequently, 20ml of

MTT solution was added to each well and treated as

the above in vitro cytotoxicity assay.

Selectivity of 5-Fu-PNA conjugate

For assessment of the cytotoxic selectivity of the

conjugate, the normal Chang cells were used. The

single cell suspension of Chang cells was obtained and

the cell number was adjusted to 4 £ 104 cells/ml with

DMEM medium. A measure of 100ml of cell

suspension was seeded into each well in a flat-bottom

96-well plate. When the cells were adhered to the

plate, the free drug and the active conjugate with

equivalent amounts of 5-Fu were added at gradually

increased concentrations and maintained for 48 h at

378C. Then 20ml of MTT solution was added and

treated the same as the in vitro cytotoxicity assay.

Results

Preparation and characteristics of 5-Fu-PNA conjugate

To explore the potential of PNA as a carrier molecule for

targeted drug delivery of 5-Fu to colorectal cancer, the

cytostatic agent was attached covalently to the protein

through EDC/NHS by two-step zero-length cross-

linking. First, the drug was converted to the correspond-

ing carboxylic-acid derivative. The object was obtained

with a yield of 76%. As indicated by 1H-NMR (DMSO-

d6) d 13.21 (1H,br,COOH), 11.92(1H,s,N3-H),

8.08(1H,d,JH–F ¼ 6:8;C6-H), 4.36 (2H,s, N1-CH2),

mp 245–2478C, 5-Fu was converted to the correspond-

ing derivative of N1-substituted 5-Fu acetate.

To avoid cross-linking of PNA molecules by

EDC, the N1-substituted 5-Fu acetate was reacted

with EDC/NHS to form an active ester intermedi-

ate (Grabarek and Gergely 1990) prior to coupling.

Afterwards, the drug was conjugated to accessible

amino-residues of PNA forming an amide-bond to

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Page 4: Lectin-mediated cytotoxicity and specificity of 5-fluorouracil conjugated with peanut agglutinin (5-Fu-PNA)               in vitro

get 5-Fu-PNA conjugate. The result of SDS-PAGE

confirmed the covalent attachment of 5-Fu to PNA.

On the lane of conjugate, there was a new band

with a little higher molecular weight than that of

PNA (Figure 1), which migrated to a distance

corresponding to molecular weight of 33,

323 ^ 424 relative to markers of 116.66, 66.2, 45,

35, 25, 18.4 and 14.4 kDa. The number of moles

5-Fu/mol PNA resulted by the new band was

14.85, which is similar to the result obtained by

TNBS method.

The affinity of the conjugate was tested by

haemagglutination test due to the agglutination of

the thymus cells from mice C57BL/6 by PNA (Gu and

Li 1983). The active conjugate was obtained with

D-gal protection. Titre value for the active conjugate

was in the same order as that obtained for PNA test

solution thus confirming lectin’s activity. The

obtained conjugate without D-gal protection was

non-active, which exhibited negative results as early

as the first dilution. The drug/lectin molar ratio

determined by TNBS of the active conjugate and the

non-active conjugate were 38.42 and 62.13%,

respectively.

In vitro cytotoxic effect of conjugate on LoVo cells

In order to provide comparability of the obtained data,

concentrations of 5-Fu of the active conjugate applied

to MTT test were equivalent to that of the free drug.

All results are expressed as % cell survival of the

control (untreated) cells over time. Results presented

in Table I and Figure 2 show the survival cells treated

by the free drug and the active conjugate at different

concentrations for 48 and 120 h, respectively. It could

be observed that there was a specific time- and

concentration-dependent antiproliferative effect of the

free drug and the active conjugate. Little toxic effect

on the cells was observed exposing the cells to the

active conjugate for 24 h. However, the cell cytotox-

icity was observed with time going on (Figure 2).

Specificity of 5-Fu-PNA conjugate

The cytotoxicity of PNA, the active conjugate and the

non-active conjugate were all tested on LoVo cells to

identify the specificity of the active conjugate. As the

concentration went up to 60mg/ml of PNA, no

significant inhibition effects were observed in the cells

either treated with PNA alone or the non-active

conjugates. On the other hand, the active conjugate

exhibited high cytotoxicity to the cells (see Figures 3

and 4). Therefore, the maintenance of affinity is of

great importance for the conjugate to exhibit its

cytotoxic effect. Only when lectin retained its affinity

did the conjugate inhibit the growth of cancer cells.

Cytotoxic selectivity of 5-Fu-PNA conjugate

In the present study, PNA was chosen to be a targeting

carrier to reduce the side effect of 5-Fu to normal

Figure 1. Vertical electrophoresis of PNA and conjugates on the

polyacrylamide gels. The direction of migration was from the top.

Electrophoresis was performed in 15% acrylamide running gels and

4% stacking gels in the presence of 0.1% sodium dodecyl sulfate at

pH 8.9 at 140 V for 4 h. The gels were stained for protein with

Coomassie brilliant blue R250. The result was analysed with the

JEL-PRO software (Mediacybernetics, USA). Lane 1: PNA; Lane2:

active conjugate; Lane 3: protein Markers of 116.66 kDa, 66.2 kDa,

45 kDa, 35 kDa, 25 kDa, 18.4 kDa and 14.4 kDa.

Table I. Cytotoxic effect of the free drug and the active conjugate on LoVo cells for 120 h treatment at various concentrations.

Free drug

(concentration mm)

Conjugate

(concentration mM)

Time (h) 1 2 4 8 1 2 4 8

24 95.75 ^ 5.33 92.66 ^ 8.32 73.05 ^ 4.45 57.52 ^ 3.36 99.71 ^ 8.17 89.07 ^ 6.80 75.37 ^ 5.82 68.06 ^ 9.01

48 85.45 ^ 9.64 69.86 ^ 7.00 40.91 ^ 9.58 23.13 ^ 6.56 95.73 ^ 6.49 76.81 ^ 9.35 62.65 ^ 7.71 47.63 ^ 12.81

72 67.53 ^ 12.90 55.20 ^ 8.78 16.68 ^ 6.92 10.76 ^ 9.02 84.26 ^ 11.6 59.06 ^ 4.73 30.89 ^ 8.81 21.54 ^ 15.44

96 60.48 ^ 11.58 43.35 ^ 5.86 2.24 ^ 1.55 28.67 ^ 6.27 61.28 ^ 8.94 47.64 ^ 9.97 14.09 ^ 7.11 5.32 ^ 3.28

120 53.10 ^ 8.27 35.49 ^ 5.24 0.80 ^ 0.37 A 50.23 ^ 7.59 39.96 ^ 6.96 4.51 ^ 3.64 22.78 ^ 1.41

All the values are means of surviving LoVo cells assessed by MTT assay. Data are expressed as a % of the control values (untreated

cells)(mean ^ SD, n ¼ 3). A: results not determined.

Q. Cai & Z.-R. Zhang254

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cells. Human normal Chang cells were used to

investigate the selectivity of the active conjugate. The

cells were treated either by the free drug or the active

conjugate for 48 h at 378C. The results were indicated

in Figure 5.

Discussion

PNA, as the carrier molecule, was conjugated with

5-Fu via an amide linkage. The affinity of PNA was

preserved after its coupling to 5-Fu when D-gal was

used for protection. Without D-gal protection, PNA

will lose its affinity after cross-link reaction, which is

due to the formation of amide bonds between

carboxylic acids of N1-substituted 5-Fu acetate and

amines of PNA. However, the free amino group at C-2

of PNA is required for its affinity to bind sugar (Lotan

et al. 1975).

SDS, which is an amphipathic molecule and

protein’s strong denaturing agent, binds to most

of proteins with the same ratio of 1.4:1 and causes

proteins to assume a rod-like shape. The large negative

charge that the SDS imparts masks the protein’s

intrinsic charge so that SDS-treated proteins tend to

have identical charge-to-mass ratios and similar

shapes. Therefore, SDS-PAGE separates proteins in

order of their molecular masses because of gel

filtration effects. The relative mobilities of proteins

on such gels vary linearly with the logarithm of their

molecular masses. In addition, SDS treatment

disrupts the noncovalent interactions between

protein’s subunits so SDS-PAGE measures the

molecular masses of the subunits rather than that of

the intact protein. Since PNA has four identical

subunits, there was only one band in the lane of PNA

in SDS-PAGE figure. After 5-Fu molecules were

conjugated to part of the subunits of PNA,

the molecular weight of which was increased.

Consequently, there was a new band in the lane of

conjugate with a slightly higher molecular weight than

that of PNA (as seen from Figure 1)

For treatment both with free drug and the active

conjugate, growth inhibition was observed in the

colorectal cancer cells under tested concentrations.

The cancer cell line showed a specific concentration-

and time-dependent sensitivity to drugs. The active

conjugate had similar cell killing ability to the free

drug. The survival rate was 48.48% for cells treated

with the free drug at the concentration of 4mM for

48 h, while the active conjugate inhibited the growth of

LoVo cells with a survival rate of 62.78% at the same

condition. It seems that the conjugated drug was less

active than free drug. Following are the underlying

reasons. Biomembrane is selectively permeable, i.e. it

allows certain substrates to cross by passive diffusion

or by active transport process, while restricting the

transfer of others. The passive diffusion is mainly

responsible for small molecules, while the active

process, which involves energy and sometimes

receptors as well, is to transport some special materials

such as large molecules. The 5-Fu-PNA conjugates

are water soluble and are of high molecular weight so

that they are unable to diffuse across biomembranes.

Their uptake into cells should be a receptor-mediated

Figure 2. Cytotoxic effect of the free drug and the active conjugate

on LoVo cells (A) Concentration-dependent curves for 48 h

treatment at gradually changed concentrations. (B) Time-

dependent curves for 120 h consecutive treatment at the

concentration of 4mM of 5-Fu. The viability of LoVo cells was

assessed by MTT assay after continuous exposure to the free drug

and the active conjugate. Data are expressed as % cell survival of the

control cells (untreated cells) (mean ^ SD, n ¼ 3).

Figure 3. The inhibition effect of PNA, the active conjugate and

the non-active conjugate on LoVo cells for 48 h treatment at various

concentrations. The viability of LoVo cells was assessed by MTT

assay after continuous exposure to the free PNA and the conjugates.

Data are expressed as % cell survival of the control cells (untreated

cells) (mean ^ SD, n ¼ 3).

Lectin-mediated cytotoxicity 255

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active pathway, which could be speculated from

the result of specificity experiment. Thereby, the

relative rate of uptake into cells became the primary

factor influencing the cytotoxicity of the conjugate.

After uptake into cells, the drug needs to be released

from the conjugate and to transport across the cellular

compartment membrane such as lysosome and

endosome into the cytoplasm. Thus, the rate and

efficiency of the release and the rate of diffusion may

lead to the decreased cytotoxicity of the conjugate.

According to Figure 2B, it took 96 h for the free drug

at the concentration of 8mM to kill almost all the cells

while for the active conjugate, it took 120 h.

Compared with the free drug, there was a time-

delayed effect of the active conjugate since it took

some time for the drugs to be released from the

conjugate and to transport across the biomembrane of

cellular compartment before they began to work.

Instead of the non-active conjugate, D-gal was used

to assess the specific binding of conjugate to LoVo

cells in our preliminary experiments. However, D-gal

could inhibit the growth of LoVo cells (data not

shown). Consequently, the non-active conjugate was

chosen in the specificity binding experiment. As a

result, the carrier protein alone had little effect on the

growth of LoVo cells, which is in accordance with the

literature (Kiss et al. 1997). The active conjugate with

affinity showed parallel cell killing effect to the free

drug. Meanwhile, little inhibition was detected after

the non-active conjugate was added to the cancer cells.

Therefore, it should be lectin that mediated the

conjugate delivery into cells. It can also be inferred

that the affinity and specificity of targeting ligands play

an important role in successful cellular delivery of the

conjugate.

A good targeting delivery system should identify the

target tissue or cells from the non-target ones. In order

to examine the selectivity of the conjugate, Chang cells

were employed. In this study, 5-Fu-PNA conjugate

exhibited much higher selectivity than the free drug

since it killed colorectal cancer cells LoVo selectively

and exhibited weak effect on the growth inhibition of

the normal liver cells Chang. However, the free drug

killed both.

Although 5-Fu-PNA conjugate was less cytotoxic

than the free 5-Fu to LoVo cells in vitro, the antitumor

effect of the conjugate in vivo may be higher than that

of free drug because it has much higher selectivity,

which might be of benefits to the accumulation of

conjugates at the tumor site. Such cases were reported

in literatures on studies of monoclonal antibody-drug

conjugate (Deguchi et al. 1986, Shawler et al. 1988).

However, the antitumor effect of 5-Fu-PNA in vivo

still needs further investigation.

Conclusion

In this study, we synthesized lectin-mediated targeting

system of 5-Fu-PNA, which fulfilled the principal

requirements for targeted systems such as binding

specificity, low toxic effect on normal cells, etc. Based

upon these optimistic results, 5-Fu-PNA conjugate

may be a promising target system, which should be

further investigated in vivo for site-specific delivery to

colon carcinoma.

Acknowledgements

This work was sponsored by the National Natural Fund

for Distinguished Young Scholars (No. 39925039) of

People’s Republic of China.

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