PENGEMBANGAN TARGETED THERAPY NANOPARTICLES

INNULIN BERBASIS MICRORNA ANTI-MIMIC MIR PADA

KULTUR SEL KANKER OVARIUM SKOV3 SECARA IN VITRO

Tim Peneliti:

Dr. Drs. Muhammad Ghufron

Aulia Zuhria Rahma
Hana Fauzyyah Hanifin
Siti Anindita

DEPARTEMEN/LABORATORIUM/PUSAT KAJIAN
FAKULTAS KEDOKTERAN
UNIVERSITAS GADJAH MADA
YOGYAKARTA
 Ovarian Cancer
• Ovarian cancer is one of the the 8th deadliest
cancer for woman all over the world.

Problem : Source :
http://www.medgurus.org/ovarian-cancer-treatment-in-india/

• Late diagnostic
• No early diagnostic
• High recurrence rate Targeted therapy as
• Chemotherapy resistance solution
• Chemotherapy side effect
 Oligonucleotide Using
based therapy microRNA (miRNA)
- Non coding single strand RNA, 17-25 base
pair
- Regulate protein coding gene in post
translation by interaction with 3’Utr of mRNA
- Important on cell regulation
- Some research are developing miRNA as
targeted therapy using RNA interference
approach as antagonis miRNA or mimic RNA

Delivery methods of miRNA tumor suppressor mimics usually given by intratumoral

or by virus vector.

Not suitable for clinical usage :

- Intratumoral injection cannot be done on metastatic or deep tumour

- Virus virus showed inactivity and limitation on nucleus translocation
This research using miR 144 and miR 320c as target therapy in vivo on ovarian
cancer cells skov3 :

- AntagomiR 320c used because miR 320c is upregulated

- Mimic miR 144 used because miR is downregulated
(genomiR FK UGM, 2016)
Using 3 models of

Single
oligonucleotide
transfection

Made by Exiqon, more

stable than ordinary
Locked Nucleic single oligonucleotide,
Acid (LNA) widely used on
oligonucleotide therapy
Inulin based agent’s research
nanoparticles
 Inulin based
nanoparticle

- Nanoparticle is colloidal submicron system (<1μm) usually formed from polymer

- 7 to 70 smaller than red blood cell, could be given by intravena without embolization risk

Why inulin :

- Use to maximized RNA delivery Basicly, sisteamin as ligan

to body’s tissue conjugated on axidated
- stabilized labil component such inulin (using periodat)
as protein and peptide that will form crosslinked
- Non toxic inulin
- Could form certain stabil
conjugate

synthesis inulin conjugate

 Problem
How is the comparison of effectivity, specificity, and toxicity of anti miR and mimic miR on
cancer cell skov3 as single strand, LNA, and inulin nanoparticle sistem?

Objective
To find out the comparison of effectivity, specificity, and toxicity of anti miR and mimic miR
on cancer cell skov3 as single strand, LNA, and inulin nanoparticle sistem?
METHOD
7

INDEPENDENT VARIABLE
• Experimental design : post test only control
grup design - Concentration of anti miR 320c and
mimic miR 144
- Delivery system of micro RNA as
single nucleotide, LNA, and inulin
SUBJECT : nanoparticle

Cancer cell line skov3, collection of

LPPT UGM. This cell growth on DMEM
high Glucose (Gibco) contain Fetal
Bovine Serum (FBS) 10% (v/v) (FBS DEPENDENT VARIABLE
Qualified, Gibco, Invitrogen USA),
penisilin-Streptomisin 1% (v/v) (Gibco, Micro RNA quantification and
Invitrogen USA) dan Fungizone 0,5 % cytotoxicity effect ( single and
v/v (Gibco) combination)
 Framework
8

Skov3 culture

Transfected with 3 microRNA qRT-PCR

models of micro RNA
(antomiR and mimic
miR)
- single nucleotide
- LNA
- inulin nanoparticle MTT assay
9
skov3 culture

maintained in media contain :

 DMEM high glucose
 penicillin and streptomycin 1%
 Fungizone 1 %
 FBS 10%

 Cell harvested using tripsin-EDTA 0,25%

 Inulin based nanoparticle
Synthesis of inulin conjugate
2 g inulin dissolved in 100 ml water + 200 g periodat. Strirred in dark room
for 2 h then dialysed. Result of dialysis then freeze dryed and take 0,5 g to
dissolve on water + 1 g MES buffer + 1,25 g sisteamin. Acidity measured on
pH 5. mixture stirred for 3h the add 4 g sodiumcyanoperiodat, continue stir
for 3 day. Purified result using dialysis tubing of cellulose membrane

Syntesis of cross linked inulin conjugate

Same with above, but sisteamin replaced with inuin-sisteamin.

Determine free thiol group using UV-vis spectrofotometer

Syntesis of nanoparticle
Made by ionic gelation reacrtion of inulin conjugate and cross linked
inulin conjugate, micro RNA dissolve on polimer with pH 5,5 and
concentration 0,2% (b/v)
 Micro RNA
11
quantification
 Isolate RNA sample using miRCURY RNA kit Cell
and Plant (Cat No. 300114, Exiqon).
 cDNA synthesis use cDNA synthesis kit II, 8-64
rxns (Cat No. 203301, Exiqon)
 RNA quantification use Biorad C.1000 qPCR. Kit
reagent that used is ExiLent SYBR Green master
mix, 2.5 mL (Cat No. 203402,Exiqon), primer set
(forward dan reverse) miRNA, cDNA
 Biorad CFX 96 : Denaturation 95oC, 10 min;
Amplification 40 cycle; 95oC, 10 sec; 60oC, 1
min ramp-rate 1,6 oC/s; Optical read; melting
curve analysis choose yes. Analysis done by
Biorad CFX ManagerTM Software to get
Quantification cycle (Cq) value,
quantification curve and melting curve.
MTT assay
- 80% cell confluence harvested
and planted on 96 well plate (4000
cell/well) the incubate.
- Media discarded after 24 h, then
washed by PBS 1x 100 ul/well. Add
100 ul test solution/ well. Incubate
24 h
- Add MTT reagent 0,5 mg/ml 100
ul/well, incubate 3-4 h, live cell will
break MTT became violet
formazan crystal.
- Add stopper solution : SDS on PBS
100 uL/ well. Incubate room
temperature overnight.
- Measure viability using ELISA
reader on λ 595 nm
Thank you