ORIGINAL RESEARCH PAPER

Horseradish peroxidase-encapsulated chitosannanoparticles for enzyme-prodrug cancer therapy

Xiaodan Cao • Chao Chen • Haijun Yu •

Ping Wang

Received: 13 June 2014 / Accepted: 3 September 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Among various enzyme-based therapies,

enzyme-prodrug therapy (EPT) promises minimized

side effects in that it activates non-toxic prodrugs

locally where the enzymes are placed. The success of

such an approach requires high enzyme stability

against both structural denaturation and potential

immunogenicity. This work examines the efficiency

of nanoparticles for enzyme protection in EPT appli-

cations. Specifically, horseradish peroxidase (HRP)-

encapsulated chitosan nanoparticles (HRP-CSNP)

were constructed and examined with respect to

stability enhancement. HRP-CSNP retained enzyme

activity and had improved stability at 37 �C in the

presence of a denaturant, urea. The nanoparticles

effectively bound to the surface of human breast

cancer cell Bcap37 and led to over 80 % cell death

when applied with a prodrug indole-3-acetic acid.

Keywords Breast cancer � Chitosan nanoparticles �Enzyme-prodrug therapy � Horseradish peroxidase �Indole-3-acetic acid � Nanoparticles for cancer

therapy � Protein stabilization

Introduction

Enzymes are widely used as highly selective thera-

peutic agents for treatment of diseases including

cancer. For example, enzyme-antibody conjugates

have been explored for disease therapy by directing

enzymes to targeted tissues (Vellard 2003; Vertegel

et al. 2011). Enzyme-prodrug therapy (EPT) alterna-

tively seeks to apply enzymes that are not toxic in

themselves but can catalyze activation of prodrugs

which are also non-toxic to produce a toxic drug at

targeted locations, thereby increasing tumor selectiv-

ity and avoiding side-effects (Bagshawe 2006). The

therapeutic effects of several EPT systems have been

reported (Andrady et al. 2011). Poor stability and

potential immunogenicity, along with insufficient

binding of enzyme to targeted cells, are the critical

limiting factors for cancer therapy (Chang et al. 2014;

de Bont et al. 1997; Sharma et al. 1992). In a clinical

trial of antibody fragment A5B7-F(ab’)2-bacterial

carboxypeptidase G2 (CPG2) conjugate, human anti-

mouse antibodies (HAMA) and anti-CPG2 antibodies

were found in serum of all patients studies within

10 days after a single treatment with the conjugate.

X. Cao � C. Chen � H. Yu � P. Wang (&)

Biomedical Nanotechnology Center, State Key

Laboratory of Bioreactor Engineering, East China

University of Science and Technology, Shanghai 200237,

People’s Republic of China

e-mail: pwang11@ecust.edu.cn

X. Cao

e-mail: xdcao@ecust.edu.cn

C. Chen

e-mail: chenchao6633@126.com

H. Yu

e-mail: 597279072@qq.com

123

Biotechnol Lett

DOI 10.1007/s10529-014-1664-5

Moreover, the anti-CPG2 antibodies could lead to

CPG2 clearance from plasma and lower concentration

of CPG2 at tumor site (Sharma et al. 1992). In another

study, monoclonal antibody-human b-glucuronidase

conjugate with the prodrug of paclitaxel was examined

but the amount of cell-bound enzyme was too low to

realize sufficient activation of prodrug in 24 h (de

Bont et al. 1997).

One of the possible approaches to overcoming these

problems is to encapsulate the enzymes in nanoparticles

which may improve both enzyme binding capacity and

stability. Controlled drug release from a nanoparticle-

based delivery system offers several advantages that are

desired for EPT applications, including protecting

macromolecules such as proteins from degradation,

directing active components to specific sites, improved

interactions with targeted cells, and avoiding immune

system recognition (Singh and Lillard 2009; Zolnik et al.

2010). For example, dual-porosity hollow silica nano-

particles are effective in protecting penicillinase from

immune attack (Ortac et al. 2014). As far as enzyme

stabilization and protection are concerned, many differ-

ent nanomaterials, including liposomes (Huysmans et al.

2005), gold nanoparticles (Kumar et al. 2005) and

polymers (Dziubla et al. 2005), are effective.

Little though has been reported on using biopolymer

nanoparticle-enzyme systems for EPT applications. The

objective of this research was to examine the efficiency

of simple nanoparticle capsulation for EPT. Towards

that, the activation of prodrug, indole-3-acetic acid

(IAA), by horseradish peroxidase (HRP), which has

been reported previously with promising potentials

(Wardman 2002), was chosen as a model reaction

system, while chitosan was chosen for nanoparticle

preparation. Chitosan nanoparticles have attracted atten-

tion in biomedical areas because of its biological origin

that affords excellent biocompatibility, low toxicity, and

high capacity for chemical modification due to the

hydroxyl and amino residue groups (Katas et al. 2013).

As an example, chitosan-based nanoparticles can protect

insulin from degradation (Chen et al. 2013).

Materials and methods

Materials

Horseradish peroxidase (HRP, 150-250 units/mg

solid), IAA, low molecular weight chitosan, and

fluorescein-5-isothiocyanate (FITC) were purchased

from Sigma-Aldrich. Triphosphate (TPP) was

obtained from Adamas-beta (Shanghai, China). Pyro-

gallol and 40,6-diamidino-2-phenylindole (DAPI)

were purchased from Sangon Biotech (Shanghai,

China). 1-Ethyl-3-[3-dimethylaminopropyl] carbodi-

imide hydrochloride (EDC) and N-hydroxy-

succinimide (NHS) were purchased from Thermo

Scientific. Rhodamine phalloidin was purchased from

Cytoskeleton Inc. (Denver, Co, USA). All other

reagents and chemicals were analytical grade. Human

breast cancer cell Bcap37 was purchased from Cell

Bank of Committee on Type Culture Collection of

Chinese Academy of Sciences.

Preparation of HPR-encapsulated chitosan

nanoparticles (HRP-CSNP)

HRP-CSNP was prepared using ionic gelation method

in two steps. In the first step, HRP was conjugated to

chitosan via EDC/NHS chemistry. HRP (0.5 mg) and

50 mg EDC was added to 1 ml Milli-Q water and kept

at 4 �C for 10 min, allowing the activation of the

carboxylate groups on HRP. Subsequently, 10 mg

chitosan was dissolved in 1 % acetic acid (1 ml) at

60 �C and then its pH was adjusted to 5. The HRP and

EDC mixture was added to the above solution

followed by the addition of 40 mg NHS under constant

stirring at 4 �C for 48 h, resulting in activated HRP

crosslinked with the primary amines on chitosan. In

the subsequent step, HRP-CSNP was prepared by

ionic gelation with TPP. The HPR-conjugated chito-

san was then diluted to 1 mg/ml and filtered using

0.22 lm membrane. The above mixture (5 ml) was

stirred at 500 rpm followed by drop-wise addition of

2 ml 1 mg TPP/ml. After 30 min, the solution was

centrifuged at 16,0009g, 4 �C for 30 min to remove

excess amounts of TPP and unencapsulated HRP. The

pellets were re-suspended in Milli-Q water and the

supernatant was collected to measure the amount of

unentrapped HRP. The process was repeated twice. To

determine cellular binding of nanoparticle, FITC-

labeled CSNP were prepared following a procedure as

reported previously (Huang et al. 2004).

Characterization of HRP-CSNP

The particle size and zeta potential of particles were

measured using Zetasizer Nano ZS (Malvern

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123

ZEN3600). The electron micrographs were taken

using a transmission electron microscope. The sus-

pension of chitosan nanoparticle (CSNP) and HRP-

CSNP were sonicated for 3 min, 10 ll was then

placed on a carbon-coated copper TEM grid. The

sample was negatively stained with 2 % (w/v)

phosphotungstic acid for 10 min. After the samples

were dried, the images were taken. The encapsulation

efficiency of HRP-CSNP was measured by monitor-

ing the concentration of HRP in the aqueous solution

before and after encapsulation using BCA protein

assay kit, with the assumption that the difference was

entirely encapsulated in the nanoparticles. The

encapsulation efficiency of the HRP-CSNP was

defined as the amount of HRP in the HRP-CSNP

compared to the total amount of HRP used. The

enzyme loading was defined as the amount of HRP

per HRP-CSNP weight.

HRP activity assay

The activity of free HRP or HRP-CSNP was deter-

mined according to the Sigma-Aldrich’s instruction

using pyrogallol and H2O2 as substrates.

Cellular binding study

Bcap37 cell (5 9 104 cells/ml) was seeded in 6-well

plate and grown in Dulbecco’s modified Eagle’s

medium (DMEM) (Gibco) with 10 % (v/v) fetal

bovine serum, 100 U penicillin/ml, and 100 lg

streptomycin/ml at 37 �C with 5 % (v/v) CO2 over-

night. The cells were then incubated with 30 lg FITC-

labeled CSNP/ml for different times at 37 �C. The cell

images were taken by a confocal microscope and

analyzed using Nikon NIS-Elements.

Cellular cytotoxicity studies

Bcap37 cells (5 9 105 cells/ml) were seeded in

96-well plates overnight. The cytotoxicity assay was

performed after cells were treated with 1.2 lg free

HRP/ml, CSNP and HRP-CSNP in the presence of

100 lM IAA for 36 h. The dose of HRP-CSNP with a

total activity equal to 1.2 lg free HRP/ml was used.

Untreated cells served as control for the experiment.

After the treatment, the cell viability was determined

by MTT assay.

Results

Preparation and characterization of HPR-

encapsulated chitosan nanoparticle (HRP-CSNP)

A schematic illustration of the preparation procedure of

HRP-CSNP is shown in Fig. 1a. HRP was first conju-

gated with chitosan chemically. The chitosan-conju-

gated HRP was then entrapped into nanoparticles

following a particle formation procedure by manipulat-

ing ionic interaction between positively charged chitosan

and negatively charged TPP. TEM analysis showed that

HRP-CSNP possessed a smooth and distinct spherical

+EDC, NHS

pH 5.0

TPP

Chitosan

HRP

TPP

OH

O

NH2

NH2

NH2

NH2

NH2

NH2

NH2

NHa

b

c

O

Fig. 1 a Procedure and mechanism for preparation of HRP-

encapsulated chitosan nanoparticle (HRP-CSNP). HRP was first

conjugated to chitosan via EDC/NHS chemistry. HRP-CSNP

was then prepared by ionic gelation with TPP. b Transmission

electron microscope image of HRP-CSNP. Bar represents

50 nm. c The average size of HRP-CSNP measured by Zetasizer

Nano ZS

Biotechnol Lett

123

shape and relatively uniformly dispersed (Fig. 1b).

Particle-size analysis revealed that chitosan nanoparti-

cles without the presence of conjugated HRP had an

average diameter of 301 ± 17 nm. When HRP was

encapsulated, the particle size increased to 334 ± 3 nm

(Fig. 1c) and the zeta potential slightly decreased (from

44 to 43 mV). The HRP encapsulation efficiency was

37.3 %, with a typical enzyme loading of 16.5 % (w/w).

Activity and stability of HRP-CSNP

The specific peroxidase activity of HRP-CSNP was

measured following a standard activity assay proce-

dure. The enzyme maintained an apparent activity of

65.7 U/mg protein, which was 42 % of the activity of

free HRP (155.8 U/mg protein).

The stability of HRP-CSNP was determined by

measuring the activity of encapsulated HRP at 37 �C.

89 % of original activity of HRP-CSNP was retained

after 20 days and 58 % of the original activity was

preserved after 50 days (Fig. 2a). In comparison, free

HRP retained only 27 % of original activity at 37 �C

over 50 days (Fig. 2a). The enzyme stability against

chemical denaturation (6 M urea) was also investi-

gated. After 60 min incubation, HRP-CSNP main-

tained 64 % of the original activity whereas free HRP

only remained 36 % of its original activity (Fig. 2b)

Cellular binding and uptake of FITC-labeled

CSNP

Binding of FITC-labeled CSNP to Bcap37 cells was

demonstrated qualitatively by confocal microscopy.

As shown in Fig. 3a, sporadic green fluorescence was

detected on the cell surface after 2 h incubation with

nanoparticles. The increased fluorescence activity was

observed over time (Fig. 3b) and eventual uptake of

CSNP by cells was found after 36 h treatment

(Fig. 3c). Three-dimensional image of cells exposed

to FITC-labeled CSNP for 24 h (Fig. 3d) and 36 h

(Fig. 3e) were taken and further confirmed that FITC-

labeled CSNP was adhered to the cell surface and

eventual internalized by the Bcap37 cells.

In vitro cytotoxicity assay

The application of HRP-CSNP for EPT was explored

using human breast cancer cell line Bcap37 as a

model. The cytotoxicity assay was analyzed after cells

treated with free HRP and HRP-CSNP in the presence

of 100 lM IAA for 36 h by MTT assay (Fig. 4). The

combined application of HRP-CSNP and IAA showed

similar cytotoxicity as the combination of free HRP

and IAA, observing less than 20 % of cell viability.

When the cells incubated with HRP, IAA or HRP-

CSNP alone, slight cytotoxicity was found.

Fig. 2 The stability of free HRP (open circle) and HRP-CSNP

(open square). Scale in the figure: 100 % HRP activity was

defined for original enzyme sample (which has a specific activity

of 155.8 U/mg protein), while 100 % HRP-CSNP activity

represented that of freshly prepared samples (with specific activity

of 65.7 U/mg protein). Error bars indicate standard deviations of

data from three independent replication experiments. a The

storage stability of free HRP and HRP-CSNP at 37 �C. To

evaluate the long-term stability of free HRP and HRP-CSNP, free

HRP or HRP-CSNP was incubated at 37 �C and samples were

removed at specific time points for the measurement of residual

HRP activity using pyrogallol and H2O2 as substrates. Briefly,

100 ll HRP or HRP-CSNP suspension was added into 2.9 ml

reaction solution [0.027 % (w/w) H2O2, 0.5 % (w/v) pyrogallol,

14 mM potassium phosphate, pH 6.0] and the increase in

absorbance at 420 nm was recorded for approx. 5 min. b The

stability of free HRP and HRP-CSNP against denaturant. Free

HRP or HRP-CSNP was dispersed in 6 M urea at 37 �C for 10, 30,

and 60 min. The residual peroxidase activity was measured

Biotechnol Lett

123

Discussion

Since both the enzyme and chitosan are charged,

electrostatic interactions between them can affect the

efficiency of enzyme encapsulation in a significant

way. CSNP loaded with proteins can be prepared by

ionic crosslinking (Jarudilokkul et al. 2011), and

Zubareva et al. (2013) found that the formation

between chitosan nanogels and peptide/proteins is

driven mostly by electrostatic interactions. Because of

the cationic properties of chitosan, chitosan-protein

complexes can be formed by electrostatic interactions

if the protein is oppositely charged. In this work, since

HRP (with an IP as 7.2) is positively charged in

Fig. 3 Cellular binding of FITC-labeled CSNP. Bcap37 cells

were incubated with 30 lg FITC-labeled CSNP/ml for 2 h (a),

24 h (b), and 36 h (c) at 37 �C. Cells were then washed several

times with PBS and followed by fixation with 4 % (w/v) para-

formaldehyde for 30 min and permeabilization using 0.2 %

Triton X-100 for 10 min. Afterwards, the cells were washed

with PBS again and Rhodamine Phalloidin (70 nM) was used to

stain the filamentous actin skeleton for 30 min. For nuclei

staining, DAPI (1 lg/ml) was used and incubated for 20 min.

The cells were imaged by confocal microscope. Three-

dimensional image of cells treated with FITC-labeled CSNP

for 24 h (d) and 36 h (e) were also taken. Scale bars = 20 lm

Biotechnol Lett

123

chitosan solution (pH 5.0) (Amidi et al. 2010), HRP

was first conjugated chemically to chitosan via EDC/

NHS bonding, and HRP-CSNP nanoparticles were

then formed by addition of the low molecular weight

anionic crosslinker tripolyphophate (TPP) (Fig. 1a).

Although the encapsulation efficiency of HRP

(37.3 %) was not very high when compared to that

of other chitosan-anionic protein complexes prepared

by the ionic crosslinking method, it effectively

improved encapsulation efficiency in comparison to

the capsulation of another positively charged enzyme,

lysozyme, which showed only 10 % encapsulation

(Piras et al. 2014).

After HRP-CSNP was formed, the zeta potential

was slightly decreased when compared to that of

CSNP. A similar trend was also found by attaching

anti-HER2 antibody to CSNP via EDC/NHS chemis-

try (Arya et al. 2011). The amine groups of chitosan

were covalently coupled with the carboxylic groups of

HRP or anti-HER2 antibody, resulting in decrease of

amine group on the surface of CSNP which accounts

for the lower zeta potential. Another interesting

phenomenon was the discrepancy in the size of

HRP-CSNP measured by Zetasizer Nano ZS and

TEM (Fig. 1b, c). This may because Zetasizer Nano

ZS measures the size of nanoparticle in aqueous media

where the HRP-CSNP swell, while TEM measures in

dry state where the particles tend to shrink (Fan et al.

2012).

The decreased specific enzyme activity of HRP-

CSNP may be partially caused by the chemical

modification of the enzyme, in addition to mass

transfer resistance considerations (Bindhu and Abra-

ham 2003). The mass transfer effect on enzyme

activity is a common observation for most immobi-

lized enzymes, including those in nanoparticles

(Chang et al. 2014; Karimi et al. 2013).

HRP in the CSNP exhibited improved properties

including storage stability and stability against dena-

turing environment (Fig. 2), as had been expected, and

this agreed well with other previously reported results

(Zhao et al. 2011).

FITC-labeled CSNP can bind to the cell surface

through a slow process in which the fluorescent

activity increased eventually with incubation time

(Fig. 3a, b). This could be due to the electrostatic

interaction between the positively-charged CSNP and

negatively-charged cell surface (Shin et al. 2013).

However, over a 24 h, the fluorescent nanoparticles

remained extracellular (Fig. 3b, d). Cellular uptake of

nanoparticles could be affected by the concentration of

nanoparticles, temperature, cell type, and physico-

chemical properties of nanoparticles such as particle

size, surface charge, and molecular weight (Hoemann

et al. 2013; Huang et al. 2002; Yue et al. 2011). Serum

proteins could also adsorb on the surface of nanopar-

ticles which influences the surface properties and

reduces cellular uptake in non-phagocytic cells (Baier

et al. 2011; Frohlich 2012; Kralj et al. 2012; Nafee

et al. 2009; Patel et al. 2010). In order to mimic

nanoparticles given into the blood stream and keep

consistence with subsequent cell experiment, relative

low concentration of FITC-labeled CSNP (30 lg/ml)

was added into the serum containing medium, which

might lead to slower cellular uptake of nanoparticles

after 36 h treatment (Fig. 3c and 3e) in this study.

The enzyme/prodrug pair, HRP/IAA, produces free

radicals and toxic 3-methylene-2-oxindole, which can

cause membrane lipid peroxidation and DNA damage,

resulting in cell death (Greco et al. 2002). When

applied HRP-CSNP in EPT, the result showed that

HRP-CSNP incubated with IAA could also lead to cell

Fig. 4 Cytotoxicity of free HRP and HRP-CSNP in presence of

IAA on Bcap37 cell by MTT assay. Cells were treated by 1.2 lg

HRP/ml, 100 lM IAA, HRP-CSNP, the combination of HRP

and IAA, and HRP-CSNP with IAA at 37 �C for 36 h. Cell

without treatment was used as control. 10 ll of MTT (5 mg/ml)

was added to the wells for 4 h at 37 �C. The medium was

subsequently removed and 150 ll DMSO was added followed

by incubation for 10 min. The absorbance of the suspension was

measured at 490 nm using the plate reader. The viability was

calculated by comparing the absorbance value of test sample to

the absorbance value of control sample

Biotechnol Lett

123

death (Fig. 4). Furthermore, compare to free HRP,

HRP-CSNP revealed longer storage stability at 37 �C

and improved stability against denaturant, urea

(Fig. 2), that combines with effective binding to the

cell surface (Fig. 3), suggesting it would allow

accumulation of sufficient enzyme and extended

lifetime of HRP-CSNP on surface of targeted cells.

All of these features of HRP-CSNP can be very

advantageous for EPT applications.

Conclusion

HRP-encapsulated chitosan nanoparticles were suc-

cessfully constructed and shown effectively stabilized

HRP. The HRP-CSNP retained high enzyme activity,

and could effectively bind to the cell surfaces,

promising sufficient enzyme activity and lifetime

desired for EPT applications.

Acknowledgments This work was supported by seed grant for

cultivating and interdisciplinary research of Chinese Education

Ministry, National Natural Science Foundation of China

(21303050), China Postdoctoral Science Foundation Grant

(2013M540341), and National ‘‘Thousand Talents Program’’

of China.

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