Saudi Pharmaceutical Journal (2016) 24, 250–253

King Saud University

Saudi Pharmaceutical Journal

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Study on fluorouracil–chitosan nanoparticle

preparation and its antitumor effect
Gaimin Chen a, Rudong Gong b,*

a
Pharmacy Department, Nanyang Medical College, Nanyang 473061, China
b
Public Teaching Department, Nanyang Medical College, Nanyang 473061, China

Available online 23 April 2016

KEYWORDS Abstract To successfully prepare fluorouracil–chitosan nanoparticles, and further analyze its
Fluorouracil; anti-tumor activity mechanism, this paper makes a comprehensive study of existing preparation
Chitosan; prescription and makes a detailed analysis of fluorouracil–chitosan in vitro release and pharmaco-
Nanoparticles; dynamic behavior of animals. Two-step synthesis method is adopted to prepare 5-FU–CS–mPEG
Preparation method; prodrugs, and infrared, 1H NMR and differential thermal analysis are adopted to analyze
Antitumor effect characterization synthetic products of prepared drugs. To ensure clinical efficacy of prepared drugs,
UV spectrophotometry is adopted for determination of drug loading capacity of prepared drugs,
transmission electron microscopy is adopted to observe the appearance, dynamic dialysis method
is used to observe in vitro drug release of prepared drugs and fitting of various release models is
done. Anti-tumor effect is studied via level of animal pharmacodynamics. After the end of the
experiment, tumor inhibition rate, spleen index and thymus index of drugs are calculated. Experi-
mental results show that the prepared drugs are qualified in terms of regular shape, dispersion, drug
content, etc. Animal pharmacodynamics experiments have shown that concentration level of drug
loading capacity of prepared drugs has a direct impact on anti-tumor rate. The higher the concen-
tration, the higher the anti-tumor rate. Results of pathological tissue sections of mice show that the
prepared drugs cause varying degrees of damage to receptor cells, resulting in cell necrosis or
apoptosis problem. It can thus be concluded that ion gel method is an effective method to prepare
drug-loading nanoparticles, with prepared nanoparticles evenly distributed in regular shape which
demonstrate good slow-release characteristics in receptor vitro and vivo. At the same time, after
completion of drug preparation, relatively strong anti-tumor activity can be generated for the
receptor, so this mode of preparation enjoys broad prospects for development.
Ó 2016 Production and Hosting by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Corresponding author. 1. Introduction

E-mail address: gongrdong@sina.com (R. Gong).
Peer review under responsibility of King Saud University. In recent years, constant social and economic development
results in people’s accelerating pace of life. Meanwhile, envi-
ronmental problems deteriorate (He et al., 2008). As a result,
global cancer incidence and mortality rates stay a high level,
Production and hosting by Elsevier posing a serious threat to people’s health, wherein, liver

http://dx.doi.org/10.1016/j.jsps.2016.04.008
1319-0164 Ó 2016 Production and Hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Study on nanoparticle preparation and its effect 251

cancer, stomach cancer, esophageal cancer and colorectal can- system; its shape is mostly solid colloidal particles with diam-
cer and other gastrointestinal cancers have a high incidence eter of 10–1000 nm (Li et al., 2012a,b). Nanoparticles have sig-
(Chao and Zhang, 2012). Currently, chemotherapy is one of nificant medicinal property transport advantages, so
the primary means for treatment of cancer, but its treatment preparation of nanoparticles has always been the focus of
effect is subject to drug side effects and drug resistance. In research questions. With constant progress and development
recent years, with the blend of molecular biology, molecular of chemical preparation level, preparation mode of nanoparti-
pharmacology, polymer materials, thermal chemistry and cles demonstrates significant diversification characteristics. In
other subjects, the researchers have developed controlled this paper, preparation effect of fluorouracil–chitosan
release preparations and targeting preparation which can be nanoparticles with ion gel method is deeply analyzed, and its
intelligently controlled according to tumor site characteristics inhibitory effect for cancer tumors is explored.
(Wang et al., 2010; Li et al., 2012a,b; He et al., 2011). Chitosan
(shown as Fig. 1) (CS), the product of deacetylation of chitin, 2. Materials and methods
is the only alkaline polysaccharide in nature. With broad range
of sources, good biodegradability, biocompatibility and low 2.1. Fluorouracil–chitosan preparation
toxicity, it is widely used in such aspects as pharmaceutical,
textile, environmental monitoring and tissue repair. But inter-
Chitosan (molecular weight 5.0
104), 5-fluorouracil, poly-
molecular and intramolecular hydrogen-bond interaction
ethylene glycol monomethyl ether 5000 (mPEG), 1-ethyl-3-(3
reduces its solubility, only partially soluble in acid, such as
-dimethylaminopropylamine) carbodiimide salt, silicate
acetic acid, hydrochloric acid, methane sulfonic acid. In order
(EDC s_ HCl), N-hydroxysuccinimide (NHS), succinic anhy-
to make excellent the properties of chitosan that are benefit for
dride, 4-dimethylaminopyridine (DMAP), N,N-dimethylf-
more cancer patients, chitosan must be modified. 5-Fluo-
ormamide (DMF), anhydrous ether, chloroacetic acid, methyl
rouracil (shown as Fig. 2) (5-Fu) is an anticancer drug of
orange/phenolphthalein, dialysis bags, and deuterated reagent.
DNA synthesis cycle. As its cells enjoy relatively strong
Acid/ alkaline buret, 8LGJ-18A freeze-drying machine,
destruction characteristics, it is widely used in the treatment
WQF-410 Fourier transform spectrometer, 500 MHz fully dig-
of a variety of solid tumors such as colorectal cancer, stomach
ital superconducting NMR spectrometer, UV-1700 UV spec-
cancer, breast cancer in clinics. However, metabolic rate of the
trophotometer, DF-101S heat collection constant
drug is relatively fast, which easily leads to problems such as
temperature magnetic stirrer, constant temperature magnetic
gastrointestinal reactions and poor selectivity, so these drugs
stirrer, differential scanning calorimeter.
are not very widespread in practice. To enhance anti-cancer
Determination of degree of deacetylation of chitosan: (1)
effect and reduce toxicity, over the years, people have done a
Determine degree of deacetylation of chitosan; (2) demarcate
lot of chemical modification work on 5-FU, to effectively
acid or alkaline solution with primary standard substance
reduce side effects of these drugs (Xu et al., 2014). Nanoparti-
anhydrous Na2CO3; and (3) Calculate the degree of deacetyla-
cles (NP) is ultra fine particle decentralized administration sys-
tion: formula is as follows:
tem formed by aggregate, poly charge of natural or synthetic
polymer materials, which belongs to colloid administration ð–NH2 Þ% ¼ f½ðC1 V1  C2 V2 Þ
0:016=Gg100%
DD ¼ ½ð–NH2 Þ%=9:94%100%
wherein C1 represents concentration of HCl, V1 represents
HCl volume needed, G represents mass of chitosan, C2 repre-
sents concentration of NaOH standard solution, and V2 repre-
sents volume of NaOH.
Preparation of polyethylene glycol monomethyl ether chi-
tosan, with synthetic method is shown in Fig. 3 below (Yan
et al., 2011a,b).
Structural characterization of polyethylene glycol mono-
methyl ether chitosan of 5-fluorouridine conjugate: (1) Weigh
1–2 mg sample after drying treatment with infrared spec-
Figure 1 Chemical structure of chitosan. trophotometry (FT-IR), after mixed compression with KBr,
measure on infrared instrument, with scan range at
4000–400 cm1; (2) nuclear magnetic resonance spectroscopy
(1H NMR), measure 1H NMR of substance with AVANCE
III 500 MHz NMR instrument, measuring solvent of 5-FUA
is DMSO-d6, measuring solvent of mPEG is D2O, and mea-
suring solvent of other substances is mixed solution of deuter-
ated water and deuterated hydrochloric acid (D2O/DCl) (Yan
et al., 2012). Degree of substitution of mPEG on chitosan is
calculated with integration of characteristic peaks in 1H
NMR. (3) Differential thermal analysis method employs Per-
kin ElmerDSC4000 for determination: Weigh 5 mg substance
for die determination, nitrogen flow rate: 20 ml/min, measur-
Figure 2 Chemical structure of 5-fluorouracil. ing range: 20–300 °C.
252 G. Chen, R. Gong

Figure 3 Preparation path of polyethylene glycol monomethyl ether chitosan.

2.2. Preparation of nanoparticles Abdomen of mice passaged to 10 d received 75% alco-

hol disinfection. Then extract milky ascites with 5 ml
5-FU–CS–mPEG, mPEG–CS, sodium polyphosphate, phos- syringe under sterile conditions, blend and inoculate to
phate buffer. armpit skin of right forelimb of mice with 1 ml syringe
SHZ-82 constant temperature oscillator, LGJ-18A freeze- under 0.2 ml/one. Conduct experiment after 3 d.
drying machine, laser particle size analyzer, KQ5200DB (2) Conduct animal grouping and administration. Kunming
CNC ultrasonic cleaner, and JEM-2010HR TEM. mice with H22 solid tumor successfully established (i.e.,
Ion induction method, with its simple, gentle features, mice with palpable solid tumor block under armpit)
becomes the most popular method for preparation of chitosan were randomly divided into six groups, half male and
nanoparticles, which takes advantage of sodium tripolyphos- half female, 20 in each group. All groups were intraperi-
phate (TPP) with non-toxic side effects for ion induction of toneally administered once every other day, with total
chitosan and derivatives to prepare chitosan nanoparticles dosage of 5 times; negative control group: normal saline,
(Yan et al., 2011a,b). Sodium tripolyphosphate contains positive control group: 5-fluorouracil solution
multiple-PO-Na+ groups, while chitosan dissolved in acetic (25 mg kg1 d1); blank vector group: mPEG–CS
acid and derivative molecular chain contains NH+ nanoparticles; experimental group: 5-FU–CS–mPEG
3 structure,
positive and negative charges between the two interacts, which nanoparticles group, divided into groups of high,
triggers intermolecular and intramolecular cross-linking and medium and low concentrations, with 5-FU concentra-
then produces nanoparticles. tion respectively at 50 mg kg1 d1, 25 mg kg1 d1,
Weigh appropriate amount of 5-FU–CS–mPEG, to be dis- 12.5 mg kg1 d1. During the period, regularly observe
solved in 1% acetic acid solution, perform magnetic stirring activities of tumor-bearing mice and make records.
until completely dissolved, adjust the pH (range 4–6) with
0.1 mol/L NaOH; under magnetic stirring, add sodium Tumor volume was calculated while tumor weight was
tripolyphosphate (TPP) solution filtered with 0.22 lm mem- weighed and tumor inhibition rate was calculated.
brane into chitosan derivative solution in drops, stir and allow Tumor inhibition rate = (tumor weight of saline group 
a period of reaction, and observe appearance of reaction tumor weight of experimental group)/tumor weight of saline
system. Centrifuge the resulting suspension at high speed and group
100%.
low temperature, discard the supernatant, add cryoprotectant, Observe apoptosis and necrotic conditions of mice
freeze and obtain drug-loaded nanoparticles. Determination of histopathologic slide.
drug loading capacity: nanoparticles drug loading
capacity = fluorouracil content/total mass of drug-loaded 2.4. Statistical methods
nanoparticles  100%.
Use SPSS 19.0 statistical software for data analysis and
2.3. Animal pharmacodynamics experiment comparison.

BL-220H electronic scale, 1 ml, 5 ml disposable syringe, H-88 3. Results

thermostat water bath, optical microscope, and vacuum dryer.
5-Fluorouracil, mPEG–CS nanoparticles, 5-FU–CS– Average particle size of drug-loaded nanoparticles (5-FU–CS–
mPEG nanoparticles, 0.9% saline, and formaldehyde solution. mPEG) is respectively 169.2 nm and 259.8 nm, and Zeta
120 healthy adult mice of clean grade, half and half of male potentials are respectively +42.55 mv and +39.27 mv; in elec-
and female weigh 18–22 g. Also, mice H22 hepatoma cell lines tron microscopy observation, nanoparticles are in regular
need to be bought. shapes with good dispersion; in vitro release test found that
drug-loaded nanoparticles show certain release effect in
(1) Establish transplanted H22 hepatoma solid tumor pH = 5.5, pH = 7.2 buffer.
model. Inoculate H22 cells with good growth after Animal pharmacodynamic experiments have shown that
recovery to abdominal cavity of Kunming mice. After tumor inhibition rates of high, medium, low concentration
5 d of intraperitoneal subculture, mice abdomen swelled group of drug-loaded nanoparticles are respectively 62.05%,
slightly. By 10 d, mice abdomen significantly mustered. 48.79% and 36.14%. Compared with negative control group,
Study on nanoparticle preparation and its effect 253

there is significant difference (P < 0.05), and tumor inhibition He, L.Q., Zhu, L., Yang, Y., 2008. Preparation and in vitro properties
rate of high dose group is comparable with positive control of folate-conjugated 5-fluorouracil chitosan nanoparticles. J.
group (5-FU) (P > 0.05); histopathologic slide shows that Guangdong Pharm. Univ. 6, 565–568.
cells of administration group are with different degrees of Li, Q., Zhuang, C.J., Cheng, M.R., 2012a. Experimental study on
galactosyl chitosan/5-fluorouracil nanoparticles’ inhibition of mice
necrosis and apoptosis.
orthotopic liver cancer transplantation model. Prog. Mod. Biomed.
35, 6819–6823.
4. Discussion and conclusion Li, Q., Zhuang, C.J., Cheng, M.R., 2012b. Experimental study of mice
liver cancer inhibition with galactosyl chitosan/5-fluorouracil
Drug-loaded nanoparticles prepared by ion gel method enjoy nanoparticles. Mod. J. Integr. Tradit. Chin. Western Med. 34,
3790–3793.
significant advantages, mainly uniform particle size, regular
Wang, J., Lin, Y.W., Wang, M.N., 2010. Preparation and properties of
shape and strong sustained release characteristics. The results
5-fluorouracil magnetic nanoparticles. Chin. J. Tissue Eng. Res. 47,
of animal experiments show that drug concentration exerts 8809–8813.
clear impact on anti-tumor effect. Therefore, during the course Xu, H.Z., Cheng, M.R., Wang, Y., 2014. Glycyrrhetinic acid modified
of drug treatment, disease parting of patients should be fully chitosan 5-fluorouracil’s inhibitory effect on liver cancer. Mod. J.
identified to develop reasonable therapeutic regimens. Integr. Tradit. Chin. Western Med. 21 (2281–2285), 2301.
Yan, C.Y., Gu, J.W., Huang, Z., 2011a. Preparation of lyophilized 5-
References fluorouracil-N-succinyl chitosan nanoparticles. Heilongjiang Med.
Pharm. 34, 19–20.
Yan, C.Y., Gu, J.W., Wang, X., 2011b. Discussion of in vitro release
Chao, Y.Y., Zhang, H., 2012. Sustained-release fluorouracil prepara-
mechanism of 5-fluorouracil-N-succinyl chitosan nanoparticles.
tion and biological safety after implantation. Chin. J. Tissue Eng.
Heilongjiang Med. Pharm. 34, 102–102.
Res. 21, 3959–3966.
Yan, C.Y., Gu, J.W., Cheng, Y., 2012. Impact of molecular parameter
He, B., Cheng, M.R., Wan, T., 2011. Preparation of galactosyl
on physicochemical properties of 5-fluorouracil-N-succinyl chi-
chitosan 5-fluorouracil nanoparticles and experimental study on its
tosan nanoparticles. Heilongjiang Med. Pharm. 35, 28–29.
inhibition of mice colon cancer. Chin. J. Clin. Med. 6, 738–741.