Post on 11-Jan-2016
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
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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
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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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