Stimuli-responsive polysaccharides based NPs

for controlled and sustained release of antineoplastic

drugs

Antonio Di Martino

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

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

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

Methods

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

Materials : Carriers

Chitosan (CS)

Polygalacturonic acid (PgA)

Alginic acid (Alg) Carboxy modified Iron (Fe-COOH)

CS-Alg

CS-Fe

CS-PgA

Therapeutic Therapeutic + Diagnostic

Methods : CS-Alg and CS-PgA NPs

Polyanion solution

Step I: Add the solution containing drug to polyanion solution

Drug(s) solutionDrug(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

Methods : Chitosan – Fe NPs

FeCl3aq

1) NaBH4

2) 4-COOH-C6H4-N2 OTsaqFe COOH

n

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

Methods : Characterization

Dynamic Light Scattering

Scanning Electron Microscopy

Transmission Electron Microscope

Magnetic properties

Morphology

Size

z-potentiaL

Stability

Magnetic Resonance Imaging

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

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 influenceMagnetic response

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

MRISignal suppression

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

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 !!!

Results : Release Kinetic – CS-Alg

0 20 40 60 80 100 1200

20

40

60

80

100

Time (h)

Cu

mu

lati

ve r

ele

ase

(%)

0 20 40 60 80 100 1200

20

40

60

80

100

Time (h)

Cu

mu

lati

ve r

ele

ase

(%)

0 20 40 60 80 100 1200

102030405060708090

100

Time (h)

Cu

mu

lati

ve r

ele

ase

(%)

HSpH7.4pH5.5pH2

HSpH7.4pH5.5pH2

HSpH7.4pH5.5pH2

Temperature : 37 º C

Increase pH release speed up

Reduction of Initial burst effect

Results : Release Kinetic – CS-PgA

0 20 40 60 80 100 1200

20

40

60

80

100

Time (h)

Cu

mu

lati

ve r

ele

ase

(%)

0 20 40 60 80 100 1200

20

40

60

80

100

Time (h)Cu

mu

lati

ve r

ele

ase (

%)

0 20 40 60 80 100 1200

102030405060708090

100

Time (h)

Cu

mu

lati

ve r

ele

ase

(%)

HSpH7.4pH5.5pH2

HSpH7.4pH5.5pH2

HSpH7.4pH5.5pH2

Temperature : 37 º C

Increase pH release speed up

Reduction of Initial burst effect

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

Short term perspective

Cell uptakes studies

Efficacy In vitro in different tumoral cell lines

In Vivo MRI

Results : Co-Encapsulation

TMZ + 5-FU mg/mg

TMZ + DOX mg/mg

5-FU + DOXmg/mg

CS-Alg (w/w = 2) TMZ280

5-FU220

TMZ310

DOX270

5-FU270

DOX420

CS-PgA (w/w = 2) TMZ210

5-FU340

TMZ260

DOX190

5-FU280

DOX350

CS-Fe (w/w = 5) TMZ180

5-FU270

TMZ120

DOX190

5-FU130

DOX210

pH : 5.5

pH : 7.4

TMZ + 5-FU mg/mg

TMZ + DOX mg/mg

5-FU + DOXmg/mg

CS-Alg (w/w = 2) TMZ280

5-FU220

TMZ290

DOX310

5-FU270

DOX420

CS-PgA (w/w = 2) TMZ210

5-FU340

TMZ260

DOX190

5-FU280

DOX350

CS-Fe (w/w = 5) TMZ90

5-FU110

TMZ190

DOX200

5-FU120

DOX310

Average value SD up to 10%

Method : Encapsulation Efficiency Effect of pH

UV-Vis

266 nm 5-FU

325 nm TMZ

480 nm DOX

100(%)

t

ft

D

DDEE

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)