Characterization and Prospect of Irradiated Chitosan as Nano

Complex Material to Deliver MicroRNA in Cancer Therapy

Firasti Agung Nugrahening Sumadi

*, Dian Pribadi Perkasa

, Tirta Wardana

, Ronny Martien

and

Sofia Mubarika Harjana

Pharmacy Department, University of Muhammadiyah Malang, Jalan Bendungan Sutami 188 A, Malang, East Java,

Indonesia 65145

Center for Application of Isotopes and Radiation, National Nuclear Energy Agency, Jalan Kuningan Barat, Mampang

Prapatan, Jakarta, Indonesia 12710

Department of Medicine, Universitas Jendral Soedirman, Jalan Dokter Medika, Purwokerto, Central Java, Indonesia

53122

Faculty of Pharmacy, Universitas Gadjah Mada, Jalan Sekip Utara, Senolowo, Sleman, Yogyakarta, Indonesia 55281

Department of Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada, Jalan Farmako, Senolowo,

Sleman, Yogyakarta, Indonesia

55281

Keywords: Irradiated Chitosan; Nano complex; MicroRNA

Abstract: Chitosan is odorless white powder derived from the partial deacetylation of chitin which is a polysaccharide

consisting of glucosamine and N-acetylglucosamine. Chitosan is commercially available in several types

and has molecular weights that vary between 10,000 and 1,000,000. Chitosan has positively charged basic

chain that can easily form nano complex with nucleic acid in this case including negatively charged

microRNA. MicroRNA (miRNA) has a large role in the regulation of cancer signaling tissue so that a

therapeutic approach is needed to restore the balance of dysregulated miRNA. The nature of microRNA

which is very susceptible to enzyme degradation requires a special system so that it is competent to deliver

microRNA into the cytoplasm. One of the factors that influence the efficiency of transfection of chitosan

nano complex with a nucleic acid to body cells is molecular weight. In this research, the chitosan molecular

weight reduction method was carried out to increase nano complex delivery using gamma-ray irradiation.

Furthermore, characterization was carried out to determine the irradiated chitosan molecular weight using

intrinsic viscosity then proceed with FTIR analysis to determine changes in chemical structure and applied

further by using it in nano complex formulations with microRNA 155-p, a microRNA that experienced

downregulation in ovarian cancer thus requiring mimic therapy. Results showed a decrease in chitosan

molecular weight after being irradiated from 110,188 dalton to 15,209 dalton while FTIR spectra showed a

break of the 1-4 glycoside bonds which was equivalent to the severance of the main chain of

polysaccharides. Electrophoresis results showed that irradiated chitosan was able to form nano complex

with 155-5p microRNA but transfection was not able to deliver 155-5p microRNA into the SKOV3 ovarian

cancer cells.

1 INTRODUCTION

Chitosan in the form of odorless white powder

derived from the partial deacetylation of chitin

which is a polysaccharide consisting of glucosamine

and N-acetylglucosamine. Chitosan is commercially

available in several types and has molecular weights

that vary between 10,000 and 1,000,000. The

general function of chitosan is as a coating agent,

disintegrant, film-forming agent, mucoadhesive,

tablet binder, and viscosity-enhancing agent.

Chitosan has been processed into several dosage

forms including gels, films, beads, microspheres,

tablets, and coatings for liposomes (Rowe, Sheskey,

Owen, & American Pharmacists Association, 2009).

Chitosan is a polymer that has a positive charge

that strong enough to form nano-sized complexes

with nucleic acids that have opposite charges,

Sumadi, F., Perkasa, D., Wardana, T., Martien, R. and Harjana, S.

Characterization and Prospect of Irradiated Chitosan as Nano Complex Material to Deliver MicroRNA in Cancer Therapy.

DOI: 10.5220/0009127001910196

In Proceedings of the 2nd Health Science International Conference (HSIC 2019), pages 191-196

ISBN: 978-989-758-462-6

191

including miRNA. MiRNA can experience

upregulation and downregulation in various cancer

cases (Kinose et al., 2014). MiRNA-based therapies

such as mimic miRNA (artificial miRNA) are used

to restore the function of miRNA that is lost due to

its low expression (Tyagi et al., 2016). Chitosan-

miRNA nanoparticles can increase cellular uptake

by binding to negatively charged cell membranes

and protecting miRNA from endogenous nuclear

digestion (Chen et al., 2014).

There are several factors that affect the

transfection efficiency of chitosan nanoparticles with

nucleic acids such as molecular weight, degree of

deacetylation, n/p ratio, nucleic acid concentration,

nucleic acid dose, pH of transfection media, serum

content, stability of nucleic acid and chitosan

complexes, toxicity of chitosan vector, chitosan

modification to facilitate transfection, and the type

of cell that is transfected. The smaller molecular

weight of chitosan will produce smaller chitosan-

nucleic acid complexation, but chitosan with a larger

molecular weight can bind plasmids more efficiently

(Raftery et al., 2013)

The gamma irradiation factor (γ) used in this

study to reduce the molecular weight of chitosan

also includes methods for the process of sterilizing

pharmaceutical products (Desai & Park, 2006).

However, irradiation will affect the performance of

the drug delivery system so characterization is

needed to determine its properties. Determination of

molecular weight is conducted to prove the

irradiation process can break chemical bonds

between chitosan polymers so that the molecular

weight becomes smaller.

2 METHODS

Irradiated chitosan characterization

Medical grade Chitosan (PT Biotech Surindo) was

dissolved became 1% chitosan solution using 100%

acetic acid as much as 100 ml. Chitosan 1% solution

is then irradiated with gamma-rays at a dose of 5

kGy. Then the radiation results are diluted into

0.01%, 0.05%, 0.1%, 0.15%. and 0.2% chitosan

solution. Irradiated chitosan characterization is done

by calculating intrinsic viscosity and FTIR test.

Intrinsic viscosity is then included in the sakurada

mark-houwink equation ɳ = KMα to obtain

molecular weights with K = 9.66x10-5 (dm3 / g) and

α = 0.742. FTIR testing was carried out with IR-

Prestigo - ZI Shimadzu serial A210048-02492 and

measured using the % transmittance measurement

model, resolution = 2, apolization = hap genzel, total

af.scan = 20, measurement distance = 400-4000 (cm-

1 ) (Sionkowska et al., 2013).

Chitosan nanoparticle

0,05% chitosan solution was prepared using acetate

buffer pH 5. Nanoparticles were prepared by mixing

500 ul mimic miRNA 155-5p 2 µM with 500 μl

0.05% irradiated chitosan (Martien, 2009).

Irradiated chitosan nanoparticle and transfection

40,000 SKOV3 ovarian cancer cell cultures (got

from the KALBE Stem Cell and Cancer Institute)

were planted on a 24-well plate that had been coated

with a borosilicate glass cover. Cells were

transfected with nanoparticles with miRNA that had

been labeled with FAM (green fluorescence) at 4

and 48 hours. Cell nuclei were stained with DAPI

blue fluorescence (4 ', 6-diamidino-2-phenylindole)

(Chen et al., 2014; Ji et al., 2009)

3 RESULTS AND DISCUSSION

Irradiated chitosan characterization

High molecular weight chitosan is widely used in

several biological applications. However, in many

cases, the application of this polysaccharide is

hampered due to high molecular weight which

causes low solubility in water-based media

(Czechowska-Biskup et al., 2005; Minagawa et al.,

2007). Some specific applications use chitosan

degradation products that are considered more

useful. Some degradation methods that can be used

are enzymatic, chemical or radiation. Degradation

using radiation was chosen because it is simple and

very environmentally friendly because it does not

require initiator and by product .

Irradiated chitosan is assumed to undergo cutting

off the main polysaccharide group so that it

produces a smaller molecular weight compared to

non-irradiated chitosan. Determination of molecular

weight is conducted by finding the average

molecular weight of viscosity. The results of the

relative viscosity obtained using the Ostwald

viscosity method. The result then processed to

determine the reduction viscosity and inherent

viscosity (table 1).

HSIC 2019 - The Health Science International Conference

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Table 1: Reduction viscosity and inherent viscosity of non-irradiated chitosan and 5kGy irradiated chitosan.

Concen

tratiom

(g/dL)

Flow time of

sample+solvent

(t) sec

Flow

time of

solvent

(to)

ɳr = t/to

ɳsp = ɳr

– 1

Reduction

viscosity,

red

= ɳsp

/ c

[ɳ

red

]

ln ɳr

Inherent

viscosity,

inh

=ln ɳr/c

5kGy

Irradiated

chitosan

0.0001

30.47111

29.166

1.044748

10447.48

0.043775

437.7541078

0.0005

30.19

29.166

1.035109

0.035109

70.21875

0.034507

69.01419285

0.001

31.052

29.166

1.064664

0.064664

64.66434

0.06266

62.65957125

0.0015

31.4

29.166

1.076596

0.076596

51.06402

0.073804

49.20283042

0.002

32.06

29.166

1.099225

0.099225

49.61256

0.094605

47.30274995

0.0015

30.16

29.166

1.034081

0.034081

22.72052

0.033513

22.34193057

0.002

30.92

29.166

1.060139

0.060139

30.06926

0.0584

29.19978821

Non-

irradited

chitosan

0.0001

31.66

29.166

1.085511

0.085511

855.1053

0.08205

820.5040716

0.0005

35.966

29.166

1.233148

0.233148

466.2964

0.20957

419.1408009

0.001

44.66

29.166

1.531235

0.531235

531.235

0.426075

426.0745991

0.0015

54.408

29.166

1.86546

0.86546

576.9732

0.623508

415.6717031

0.002

64.782

29.166

2.221148

1.221148

610.574

0.798024

399.0120699

Figure 1: FTIR result of (a) non-irradiated chitosan and (b) 5kGy irradiated chitosan.

Characterization and Prospect of Irradiated Chitosan as Nano Complex Material to Deliver MicroRNA in Cancer Therapy

193

Figure 2: Agarose Electrophoresis Inhibition Test (a) naked miRNA-155 5p (b) Nanoparticle Chitosan irradiated-mimic

miRNA 155-5p

Figure 3: Transfection result of irradiated chitosan-miRNA nanoparticle to SKOV3 cell culture. (a) naked miRNA;

(b) irradiated chitosan-miRNA nanoparticle after 4 hours transfection; and (c) irradiated chitosan-miRNA nanoparticle after

48 hours transfection.

The intercept results from reduction viscosity

and inherent viscosity is intrinsic viscosity (ɳ) which

is then calculated in the Mark-Houwink Sakurada

equation ɳ = KM

with the constant for chitosan K =

9.66x10-5 (dm

/ g) and

= 0.742 to obtain average

molecular weight. The result shows the average

molecular weight of non-irradiated chitosan is

110,188 Dalton while the average molecular weight

for 5 kGy irradiated chitosan is 15,209 Dalton.

FTIR method is used to identify changes in

chemical groups that occur in irradiated chitosan.

From the spectra, we obtained a strong peak

absorption in 1650 cm

-1

area which shows the

presence of carbonyl groups (fig 1a and 1b). There

are also OH groups illustrated with peaks in the

2800 cm

-1

region. The ester group (C-O) is also

found by absorption in the 1100 and 1198 cm

-1

regions. Meanwhile, phenol alcohol groups were

seen from wide absorption in the area of 3300-3400

-1

. The ether group (C-O) can be seen from the

absorption in the area of 1100 to 1300 cm

-1

. The

amino group is seen from a double peak in an area of

about 3400 cm

-1

(Silverstein et al., 2005). The

significance of irradiated and non-irradiated chitosan

spectra can be seen from the decrease in peak

absorption in the region of 1100-1300 cm

-1

Meanwhile, the uptake in the area of 1650 cm

-1

shows both maintain their aliphatic structure. The

results of the FTIR (Fourier Transform Infra-Red)

spectrophotometer between irradiated chitosan and

nonradiated chitosan showed similar peaks.

Identification of chitosan irradiation spectra that

is identical at the characteristic peaks shows that

there are no new chemical groups formed by gamma

irradiation. It can be said that gamma irradiation

does not induce cross-binding processes between

chitosan molecules. Although high energy gamma

radiation can react with chitosan groups that form

free radical groups, these high-energy groups cannot

easily interact because chitosan is at a solid level

(Desai & Park, 2006). The absorption of ionizing

radiation agents causes the localization of radical

elements in the carbon atoms C1 and C4, thus

breaking the glycoside bonds 1-4 which is

equivalent to breaking the main chain of the

polysaccharide. This can be seen clearly from the

reduction in irradiation chitosan peak spectra at 1100

-1

to 1300 cm

-1

(fig 1a and 1b) compared to

irradiated chitosan which is a picture of the presence

of C-O groups (Rosiak et al., 1992). This scheme is

compatible with the polysaccharide degradation

scheme which causes a decrease in molecular weight

in chitosan irradiation.

a b

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Chitosan nanoparticle and efficacy of miRNA

transfection

The MiR-155-5p expression is known to

experience downregulation in advanced stages of

ovarian cancer compared to early stages and

followed by upregulation of HIF1α mRNA

expression (Chasanah et al., 2016). Mimic-miRNA

is given to ovarian cancer cells to improve the

dysregulation of miR-155 5p on SKOV3.

The results of the agarose electrophoresis

inhibition test showed that mimic-miR 155-5p

trapped in irradiated chitosan (fig.2) to form

nanoparticles so that there is no free miRNA band.

This shows that nano complex can be formed

between mimic-miR-155-5p with irradiated chitosan

(Kaban et al., 2017; Martien, 2009; Wu et al., 2016).

The nanoparticle uptake test in SKOV3 cell

culture was carried out by transfecting mimic-

miRNA 155-5p nanoparticles for 2 different period,

4 hours (fig. 3b) and 48 hours (fig. 3c). The

irradiated chitosan-mimic miRNA 155-5p

nanoparticle transfection test was conducted by

observing whether there was an accumulation of

mimic-miRNA 155-5p green fluorescence around

the SKOV3 cell nucleus that had been stained with

blue dye DAPI. The nanoparticle formula showed

that at least 155-5p mimic-miRNA entered the cell

at the 4

hour of transfection and decreased at the

hour.

Particle size and charge from the surface of the

nanoparticles play an important role in the uptake of

nanoparticles to the cell and the efficiency of the

transfection system (Wu et al., 2016). Chitosan

irradiation could reduce the surface charge of the

particles due to amino group termination during the

radiation process. The results of FTIR showed the

bias of the absorption of amino groups showed in the

region 3300-3400 cm

-1

. This shows that during the

irradiation process there was an interruption of the

group which caused the chitosan to lose its

positive charge.

4 CONCLUSIONS

5kGy gamma-rays irradiated chitosan showed a

reduction in molecular weight so that the viscosity

of chitosan solution was decrease than non-

irradiated chitosan. Irradiation indicates a break in

the main polysaccharides chain of chitosan. mimic-

miRNA 155-5p nanoparticle formulation with

irradiated chitosan showed good results in the

electrophoresis test but not in transfection test.

Irradiated chitosan nanoparticles were not able to

bring mimic-miRNA into the cell. This is possible

because the irradiation process cuts off the amino

group which makes the chitosan charge more

negative and is unable to carry mimic-miRNA 155-

5p through the positively charged cell membrane.

ACKNOWLEDGMENTS

The author thanks SCI KALBE, Center for

Application of Isotopes and Radiation, National

Nuclear Energy Agency, Indonesia and also LPPT

UGM as the opportunity to conduct the research.

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