Transcript In The Name of God - University of Waterloo

By: Seyedeh Parinaz Akhlaghi
Supervisors: Professor Atyabi
Professor Dinarvand
Adviser: Dr. Ostad
Assistant: Dr. Saremi
Sep. 2009

Cancer: A leading cause of
death

We are in crisis in a fight
against cancer !
 Nanomedicine
brings
new hope!
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





Isolated from the bark of Taxus
brevifolia
Mechanism of action : unique!
Treatment of ovarian and breast
cancers
Poorly soluble in water
Taxol®: 6 mg/ml, Cremophor® EL
and ethanol
Cremophor® EL side effects :
⋇
Hypersentivity
⋇
Nephrotoxicity
⋇
Neurotoxicity
⋇
Effects on endothelial and vascular muscles
causing vasodilatation, labored breathing,
lethargy and hypotension
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Increasing antitumor efficacy while reducing systemic side effects
 Liposomes
 Micelles
 Cyclodextrin
 Synthesis of prodrugs
 Polymeric nanoparticles
 Conjugation to a stable macromolecular drug
- Albumin(Abraxane® : recurrent metastatic breast cancer)
- Globulins
- Synthetic polymers
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
Particles between 1 nm and 500 nm (very small particle size)

Narrow size distribution

Surface features for target-specific localization

Protective insulation of drug molecules to enhance stability

Feasibility of delivery of multiple therapeutic agents in a single
formulation

Longer circulation times

Increased solubility and biocompatibility
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Conventional vs. Nanoparticles in
cancer treatment
CONVENTIONAL CHEMOTHERAPY
⋇
⋇
Toxicity of drugs to normal
NANOPARTICLES IN CANCER THERAPY

A carrier for entering through
tissues
fenestrations in tumor
Short circulation half-life in
vasculature
plasma

Allowing direct cell access
⋇
Limited aqueous solubility

Delivery of high drug
⋇
Non-selectivity restricting
concentrations to the targeted
therapeutic efficacy
cancer cell

Reducing toxicity of normal
tissue
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Nontoxic
Biocompatible
Biodegradable
Mucoadhesive
 Thiolating Chitosan:
 Improving mucoadhesive properties
 Additional mucosal permeation-enhancing properties
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 Developing a polymeric drug delivery system for PTX
 Cremophor® EL-free
 Intended to be orally administered
 To achieve this aim:
PTX-loaded poly(methyl methacrylate) thiolated chitosan nanoparticles
were prepared by radical polymerization
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
Chitosan depolymerization

Measurement of molecular weight

Preparation of chitosan-glutathione conjugate and quantification

Preparation of blank and PTX-loaded core-shell nanoparticles

Characterization of nanoparticles

Physical status of PTX in the nanoparticles

Drug encapsulation efficiency and drug loading

In vitro PTX release

Mucoadhesion studies
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1.
Dissolving chitosan in 6% v/v acetic acid
2.
Adding NaNO2
3.
Setting pH up to 9.0 with NaOH 4M
4.
Filtering, washing with acetone
5.
Dialysis (acetic acid 0.1 M) twice 90 min, one over
night
6.
Freeze drying
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
GPC

Pollulane standards

PL Aquagel-OH mixed gel filtration column (300 mm × 7.5
mm internal diameter, pore size 8 µm)

Detector : refractive index signal detector (RID)

Mobile phase : 0.2 M acetic acid and 0.1 M sodium acetate

Flow rate : 4 ml/min
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*:Mw=31113 Da
NaNO2
MW
(mg/ml)
(g/mol)
Chtlow
7.0
15,482
Chtmed
2.7
31,113
Chthigh
1.6
84,308
Chitosan
Pollulane calibration curve
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1.
Adding chitosan to 1 M HCl (setting pH up to 5)
2.
Adding GSH, EDAC and NHS (resetting pH up to 5)
3.
Incubating for 15 h
4.
Dialyzing
5.
1.
5 mM HCl for 12 h
2.
5 mM HCl containing 1% NaCl for 12 h, twice
3.
1 mM HCl for 12 h, twice
Freeze drying
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 Iodine
titration
1.
Hydrating lyophilized thiolated polymers in demineralized water.
2.
Setting the pH to 2–3 with 1 M HCl
3.
Adding 500 µl of aqueous starch solution (1%)
4.
Titrating with an aqueous iodine solution until a permanent light
blue color was maintained.
2R-SH + I2→ R-S-S-R + 2 I- + 2 H+ + I2 (exc)
Complex
+amylose
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Total amount
Disulfide
of sulfhydryl
Chitosan
Free thiol
content
groups
(µmol/g)
(µmol/g)
(µmol/g)
Chtlow
646.36
552.66
93.70
Chtmed
618.85
532.17
86.68
Chthigh
598.15
529.04
69.11
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
Dissolving Cht or Cht-GSH in 0.2 M nitric acid ,in a two necked flask at
40°C, under gentle stirring and nitrogen bubbling.

Adding 0.08 M cerium (IV) ammonium nitrate (CAN)

For blank nanoparticles


Adding 0.25 ml of MMA under vigorous stirring
For PTX-loaded nanoparticles

Dissolving PTX in 0.5 ml methanol

adding 0.25 ml MMA

adding to the two necked flask at 40 °C under vigorous stirring
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
Keeping nitrogen bubbling for another 10 min

continuing the reaction at 40 °C under stirring for 30 min

Adjusting pH to 4.5 with 1 M NaOH

Dialyzing


16 μmol/L acetic acid

twice for 90 minutes and once overnight
Freeze drying
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NP
NP Efficiency
Chtlow-GSH-1%
43.05±6.46
Chtlow-GSH-2%
53.01±9.54
Chtlow-GSH-3%
56.31±7.31
Chtlow-GSH-4%
50.83±7.17
Chtlow-GSH-5%
48.05±5.29
N=3
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Scanning
electron microscopy
SEM (XL 30, Philips, Netherlands)
Chtlow-GSH-3% PTX Nanoparticles
18/32
Transmission
electron microscopy
TEM (CEM 902A, Zeiss, Germany)
Chtlow-GSH-3% PTX Nanoparticles
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
Measurement of particle size and PDI


Laser Light Scattering (Zetasizer Zs, Malvern, UK)
Determination of ζ potential

Laser Doppler Electrophoresis (Zetasizer Zs, Malvern, UK)
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Nanoparticle
Size (nm)
ζ potential (mV)
PDI
Chtlow-GSH
Chtmed-GSH
Chthigh-GSH
Chtlow
Chtmed
Chthigh
Chtlow-GSH-1%
Chtlow-GSH-2%
Chtlow-GSH-3%
Chtlow-GSH-4%
Chtlow-GSH-5%
Chtmed-GSH-1%
Chthigh-GSH-1%
Chtparent-1%
Chtlow-3%
151
188
228
142
189
225
196±30
133±19
141±28
144±9
164±23
207
230
249
132
44.1
59
47.4
36.7
38.7
39
36.3±3.4
33.5±4.1
34.1±6.0
30.1±1.8
37.8±3.6
29.1
32.6
43.2
37.1
0.066
0.08
0.19
0.408
0.055
0.21
0.065±0.017
0.443±0.381
0.069±0.054
0.137±0.020
0.200±0.038
0.043
0.104
0.39
0.062
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
Statistical data analyses were performed using statistical software
program (SPSS® 15, Microsoft).

Comparison of the data was performed using the Student’s t test with p <
0.05 as the minimal level of significance.
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Condition
Result
P value
Thiolating Cht
Does not affect size
P < 0.6
Increase in Mw
Size increases
P < 0.001
Incorporating PTX
Does not affect size
P < 0.659
Condition
Result
P value
Thiolating Cht
Increase in ζ
P < 0.05
Increase in Mw
Does not affect ζ
P < 0.519
Incorporating PTX Does not affect ζ
P < 0.132
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


Differential Scanning Calorimetry (DSC 822e, Mettler Toledo)
Flow rate : 10 ml/min
Heat rate : 10 °C/min
Chtlow-GSH-3% PTX Nanoparticles
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
Ultra centrifuging at 10,000 rpm and 4.0°C for 1 h

HPLC

Stationary phase:C18 column (25 × 0.46 cm, i.d., pore size 5 µm)

Mobile phase: acetonitrile:water (60:40, v/v)

Flow rate: 1 ml/min

Detector: UV (227 nm)
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4.5
y = 0.0704x
R² = 0.9993
4
AUC (kAU)
3.5
Conc.
(µg/ml)
AUC
0.5
60637±7540
5.0
357894±87868
10.0
676117±189714
25.0
1819901±467091
50.0
3496615±538383
3
2.5
2
1.5
1
0.5
0
0
10
20
30
40
50
60
Conc. (µg/ml)
N=9
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PTX loaded
Drug added
Encapsulation
Drug loading
nanoparticles (%)
(mg)
efficiency (%)
(%)
Chtlow-GSH-1% PTX
2.7
95.31±5.32
5.76±0.68
Chtlow-GSH-2% PTX
5.4
95.17±5.73
8.42±2.30
Chtlow-GSH-3% PTX
8.1
98.27±1.11
12.04±3.71
Chtlow-GSH-4% PTX
10.9
97.67±2.12
14.56±1.12
Chtlow-GSH-5% PTX
13.6
97.83±2.80
24.51±4.45
N=3
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
Dialysis method

PBS (pH=7.4) + 0.1%
tween 80, 37°C

HPLC
Chtlow-GSH-3% PTX Nanoparticles
N=3
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
Periodic acid/Schiff (PAS) colorimetric method

Samples + periodic acid

Incubated in 37 °C in a water bath for 2 h

Abs.(Au)
0.125
0.43±0.079
0.250
0.78±0.132
0.375
1.20±0.195
0.400
1.29±0.198
0.500
1.55±0.138
Schiff reagent was added at room temerature
After 30 min, absorbance was recorded at 555 nm
UV Absorbtion (Au)

Conc.
mg/2m
(l)
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
y = 3.208x + 0.206
R² = 0.998
0
0.1
0.2
0.3
0.4
0.5
0.6
Mucin Concentrations (mg/ml)
N=4
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
Preparation of mucin solution (0.5mg/ml)

Vortexing nanoparticles in mucin solution, shaking for 1 h in 37 °C

Ultracentrifuging the suspensions for 5 min in 12000 rpm

Analyzing the supernatant for the free amount of mucin

Applying the same procedure as for standard solutions

Thiolated>nonthiolated(p<o.oo3)
N=3
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 Preparation of PMMA-thiolated chitosan PTX-loaded nanoparticles
by radical polymerization
 Monodispersed spheres
 Size range: 130-250 nm
 Positive surface charge
 High encapsulation efficiency up to 98.27%
 Sustained in vitro release up to 10 days
 Considerable mucoadhesive properties
31/32
1.
Applying other hydrophobic anticancer drugs, as a model
2.
Evaluating the cytotoxicity of the nanoparticles on different cell
lines and comparing the IC50 with Taxol® and Abraxane ®
3.
4.
Investigations in animal tests
In vivo experiments for evaluating the oral absorption of the
nanoparticles
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