By: Seyedeh Parinaz Akhlaghi

Supervisors: Professor AtyabiProfessor Dinarvand

Adviser: Dr. OstadAssistant: 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 CHEMOTHERAPY NANOPARTICLES IN CANCER THERAPY

⋇ Toxicity of drugs to normal

tissues

⋇ Short circulation half-life in

plasma

⋇ Limited aqueous solubility

⋇ Non-selectivity restricting

therapeutic efficacy

A carrier for entering through

fenestrations in tumor

vasculature

Allowing direct cell access

Delivery of high drug

concentrations to the targeted

cancer cell

Reducing toxicity of normal

tissue

Conventional vs. Nanoparticles in cancer treatment

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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

Pollulane calibration curve11/32

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

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

5. 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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Chitosan

Total amount

of sulfhydryl

groups

(µmol/g)

Disulfide

content

(µmol/g)

Free thiol

(µmol/g)

Chtlow 646.36 552.66 93.70

Chtmed618.85 532.17 86.68

Chthigh598.15 529.04 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% 6.46±43.05

Chtlow-GSH-2% 9.54±53.01

Chtlow-GSH-3% 7.31±56.31

Chtlow-GSH-4% 7.17±50.83

Chtlow-GSH-5% 5.29±48.05

17/32N=3

Scanning electron microscopy

SEM (XL 30, Philips, Netherlands)

Chtlow-GSH-3% PTX Nanoparticles18/32

Chtlow-GSH-3% PTX Nanoparticles

Transmission electron microscopy

TEM (CEM 902A, Zeiss, Germany)

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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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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

Differential Scanning Calorimetry (DSC 822e, Mettler Toledo) Flow rate : 10 ml/min Heat rate : 10 °C/min

Chtlow-GSH-3% PTX Nanoparticles24/32

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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Conc. (µg/ml) AUC

0.5 60637±7540

5.0 357894±87868

10.0 676117±189714

25.0 1819901±467091

50.0 3496615±538383

N=9

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PTX loaded

nanoparticles(%)

Drug added

(mg)

Encapsulation

efficiency(%)

Drug loading

(%)

Chtlow-GSH-1% PTX2.7 95.31±5.32 5.76±0.68

Chtlow-GSH-2% PTX5.4 95.17±5.73 8.42±2.30

Chtlow-GSH-3% PTX8.1 98.27±1.11 12.04±3.71

Chtlow-GSH-4% PTX10.9 97.67±2.12 14.56±1.12

Chtlow-GSH-5% PTX13.6 97.83±2.80 24.51±4.45

27/32N=3

Dialysis method

PBS (pH=7.4) + 0.1%

tween 80, 37°C

HPLC

Chtlow-GSH-3% PTX Nanoparticles

28/32N=3

Periodic acid/Schiff (PAS) colorimetric method

Samples + periodic acid

Incubated in 37 °C in a water bath for 2 h

Schiff reagent was added at room temerature

After 30 min, absorbance was recorded at 555 nm

Conc.

mg/2ml((

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

29/32N=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

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

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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. Investigations in animal tests

4. In vivo experiments for evaluating the oral absorption of the

nanoparticles

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