nanoparticles, chitosan, iron nanoparticles, doxorubicin, temozolomide , 5-fluorouracil, controlled release

1. Stimuli-responsive polysaccharides
based NPs for controlled and
sustained release of antineoplastic
drugs
Antonio Di Martino

2. What are Stimuli Responsive Polymers (SRP)?
 High performance materials
 Show a sharp change in properties upon a small or modest modification in
environmental condition
 Diverse range of applications
 Optical systems
 Coating
 Diagnostic
 Tissue engineering
 Drug delivery

3. SRP : Importance in drug delivery
Blood 7.35-7.45 Stomach 1.0-3.0 Colon 7.0-7.5
6.0-6.5
5.0-6.0
4.5 -5.0
 pH in different tissue and cellular compartment
Tumor cells extracellular matrix
pH is lower than in healthy cells

4. Aims of the work
• Preparation and characterization of polysaccharides
based NPs for therapeutic and diagnostic application
• Encapsulation and co-encapsulation of three anticancer
drugs
• Effect of pH on the release kinetic

5. Methods

6. Materials : Antineoplastic drugs
Doxorubicin (DOX) Temozolomide (TMZ) 5-Fluorouracil (5-FU)
 Anti-metabolites
 Head, neck, colon, skin
 Easy to handle
 Good stability
 Side effects
 Alkylating agent
 Short plasma t ½
 Grade IV glioma
 Metastic melanoma
 Alkylating agent
 High therapeutic efficacy
 Widely used
 Side effects
 Encapsulation in nanoparticles structure to prolong and maintain
the drug concentration in the therapeutic window
 Reduction of side effects

7. Materials : Carriers
Chitosan (CS)
Polygalacturonic
acid (PgA)
Alginic acid (Alg) Carboxy modified
Iron (Fe-COOH)
CS-Alg
CS-Fe
CS-PgA
Therapeutic Therapeutic + Diagnostic

8. Methods : CS-Alg and CS-PgA NPs
Polyanion solution
Step I: Add the solution containing drug to polyanion solution
Drug(s) solution
Drug(s) + Polyanion
in solution
Step II: Add drop-wise A to B under magnetic
stirring
Drug(s) + Polyanion
in solution
(A)
CS solution
(B) Nanoparticles
in suspension

9. Methods : Chitosan – Fe NPs
 Stirring
 Ultrasound
 Temperature
O. A. Guselnikova, M. V.Gromov, A. I. Galanov, Adv. Mat. Res. 2014, 1040, 309-313.
Step I: -COOH modified Fe NPs preparation
Step II: coating and loading
CS Drug

10. Methods : Characterization
 Dynamic Light Scattering
 Scanning Electron Microscopy
 Transmission Electron Microscope
 Magnetic properties
 Morphology
 Size
 z-potentiaL
 Stability
 Magnetic Resonance Imaging

11. Method : Encapsulation and Release
 Encapsulation Efficiency
 preparation media (pH 5.5)
 physiological media (pH 7.4)
 Human Serum (HS)
 Simulated Gastric Fluid (SGF) : pH 2
 Preparation media (PM) : pH 5.5
 Physiological solution (PS) : pH 7.4
 Release kinetic ( T = 37 ºC)
 UV-Vis  Absorbance at different wavelength

13. Results : NPs characterization
CS-Alg
 d : 110 nm
 z-pot. + 35 mV
CS-PgA
 d : 130 nm
 z-pot : + 33 mV
CS-Fe
 d : 135 nm
 z-pot : + 30 mV
 After 1 month size
increase of only 20 %
 Great shelf-life
 No particular storage
conditions required
 uncoated
 coated
 d : 10 nm
 z-pot : - 25 mV
Coating influence
Magnetic response

14. Results : CS-Fe NPs - MRI
 T1 relaxation
High fat content tissues : Bright
Water filled tissues : Dark
.
 T2 relaxation
Water filled tissues : Bright
High fat content tissue : Dark
MRI
Signal suppression

15. Results : Encapsulation
CS-Alg
pH 5.5 61 % 57% 45%
pH 7.4 26% 24% 31%
CS-PgA CS-Fe
pH 5.5 52 % 64% 43%
pH 7.4 31% 37% 28%
pH 5.5 49 % 55% 31%
pH 7.4 23% 15% 12%
DOX
TMZ
5-FU
More than 50 % of encapsulation = around 400 mg/mg

16. Results : Co-Encapsulation
pH 5.5 50% 55% 46%
pH 7.4 20% 21% 18%
pH 5.5 58% 57% 59%
pH 7.4 27% 24% 35%
pH 5.5 61 % 54% 49%
pH 7.4 26% 21% 31%
TMZ 5-FU
TMZ
DOX
DOX
5-FU
+
+
+
CS-Alg CS-PgA CS-Fe
Drugs are well balanced in the system !!!

17. Results : Release Kinetic – CS-Alg
0
20
40
60
80
100
0 20 40 60 80 100 120
Cumulativerelease(%)
Time (h)
0
20
40
60
80
100
0 20 40 60 80 100 120
Cumulativerelease(%)
Time (h)
0
20
40
60
80
100
0 20 40 60 80 100 120
Cumulativerelease(%)
Time (h)
HS
pH7.4
pH5.5
pH2
HS
pH7.4
pH5.5
pH2
HS
pH7.4
pH5.5
pH2
 Temperature : 37 º C
 Increase pH release speed up
 Reduction of Initial burst effect

18. Results : Release Kinetic – CS-PgA
0
20
40
60
80
100
0 20 40 60 80 100 120
Cumulativerelease(%)
Time (h)
0
20
40
60
80
100
0 20 40 60 80 100 120
Cumulativerelease(%)
Time (h)
0
20
40
60
80
100
0 20 40 60 80 100 120
Cumulativerelease(%)
Time (h)
HS
pH7.4
pH5.5
pH2
HS
pH7.4
pH5.5
pH2
HS
pH7.4
pH5.5
pH2
 Temperature : 37 º C
 Increase pH release speed up
 Reduction of Initial burst effect

20.  Chitosan based nanoparticles for therapeutic and diagnostic application
has been prepared
 NPs present size less than 150 nm and high stability up to one month
 CS-Fe NPs show high T1 and T2 suppression
 High encapsulation efficiency ( > 50 %) for single or multiple loading
 Release rate can be increase or decrease according with pH
 Reduction of initial burst effect compared with other systems

21. Short term perspective
 Cell uptakes studies
 Efficacy In vitro in different tumoral cell lines
 In Vivo MRI

23. Results : Co-Encapsulation
TMZ + 5-FU
mg/mg
TMZ + DOX
mg/mg
5-FU + DOX
mg/mg
CS-Alg (w/w = 2) TMZ
280
5-FU
220
TMZ
310
DOX
270
5-FU
270
DOX
420
CS-PgA (w/w = 2) TMZ
210
5-FU
340
TMZ
260
DOX
190
5-FU
280
DOX
350
CS-Fe (w/w = 5) TMZ
180
5-FU
270
TMZ
120
DOX
190
5-FU
130
DOX
210
pH : 5.5
pH : 7.4
TMZ + 5-FU
mg/mg
TMZ + DOX
mg/mg
5-FU + DOX
mg/mg
CS-Alg (w/w = 2) TMZ
280
5-FU
220
TMZ
290
DOX
310
5-FU
270
DOX
420
CS-PgA (w/w = 2) TMZ
210
5-FU
340
TMZ
260
DOX
190
5-FU
280
DOX
350
CS-Fe (w/w = 5) TMZ
90
5-FU
110
TMZ
190
DOX
200
5-FU
120
DOX
310
 Average value SD up to 10%

24. Method : Encapsulation Efficiency
 Effect of pH
 UV-Vis
 266 nm 5-FU
 325 nm TMZ
 480 nm DOX
100(%) 




 

t
ft
D
DD
EE
 preparation media (pH 5.5)
 physiological media (pH 7.4)
 EE = Encapsulation Efficiency
 Dt = total amount of drug (mg/mL)
 Df = amount of free drug after encapsulation
(mg/mL)