Chitosan grafted carboxy functionalized polylactidenanoparticles for multidrugs controlled and sustained

release

Antonio Di Martino

Center of Polymer SystemsTomas Bata University in Zlin

Czech Republic

dimartino@ft.utb.cz

National ResearchTomsk Polytechnic University

Russia

Drug Delivery Systems (DDS)

Traditional drug delivery systems

Oral Injection based Inhalational/Pulmonary Transdermal

Why new delivery systems?

Targeted drug delivery Maximum efficacy with minimum side sffects Controlled drug delivery Optimize drug’s therapeutic effects, convenience and dose Enhance product life-cycle Improve patient compliance Control over all healthcare cost

Systems for the delivery of drugs to target sites of pharmacological actions

Nanoparticles as DDS

I) Dispersion or solid form with size in the range 10-700 nm

II) Various morphologies – nanospheres, nanocapsules, nanomicelles, nanoliposomes etc…

III) Drug (s) can be : dissolved, entrapped, encapsulated or attached to the nanoparticle matrix

IV) High Encapsulation Efficiency

V) Drug protection

VI) Controlled Release

VII) Decrease side effects

VIII) Reverse tumor multidrug resistance

IX) Cell Internalization

Why Nanoparticles ?

Polysaccharides based Nanoparticles

Polysaccharides have been considered as one of the most promising material for drug delivery

Various Sources : Algae , Microbial, Plants and Animals

Abundant

Low Cost

Large number of Reactive Groups

Chemical composition

Wide range of Mw

Biocompatible

Biodegradable

Low Immunogenity

Not Toxic

Chitosan Hyaluronic Acid

Alginate Cyclodextrin

DextranPectin

Chitosan (CS)

Physico-chemical properties

Molecular weight Deacetylation degree Viscosity Solubility Antibacterial Mucoadhesive Reactivity Complexation ability

Conventional formulations Compression tablets Wet granulation Gels Films Emulsions Wetting agent Coating agent Microspheres Microcapsules

Novel applications Bioadhesion Drug delivery Vaccine delivery DNA delivery

Aim of the work

Preparation and characterization of amphiphilic nanoparticles based on chitosan grafted carboxy functionalized polylactide

Encapsulation of Doxorubicin , Temozolomide, 5-Fluorouracil

Influence of environment on the release trend

Doxorubicin (DOX) Temozolomide (TMZ) 5-Fluorouracil (5-FU)

Polylactide-citric acid synthesis (PLACA)

Carboxy functionalized PLA has been obtained through direct melt polycondensationof Lactic acid (LA) and Citric acid (CA)

Methanesulfonic acid (MSA) as initiator. More effective compared to metal salts MSA is adapt to obtain low Mw PLA

Lactic acid

Citric acid

Carboxy functionalized polylactide (PLACA)

Kucharczyk et al. Journal of Applied polymer Science 2011, 122,1275-1285

CS-g-PLACA synthesis

Coupling reaction between CS amino groups and PLACA carboxy groups EDC as COOH activator

R1-NH2 +  HOOC-R2 → R1-NH-CO-R2 + H2O

Improve chitosan stability in solution Encapsulate hydrophobic and hydrophilic drugs simultaneously

Di Martino, Sedlarik, Int. J.Pharm . 2014 Oct 20, 474, 1-2, 134-45

CS-g-PLACA Nanoparticles preparation

Polyelectrolytes complexation method

Easy Fast Low cost Solvent-free Possibility to modulate size and surface charge

of nanoparticles

Dextran sulfate ( Mw 50 kDa ) as polyanion

Polymer to DS ratio (w/w) : from 0.1 to 5

TMZ – DOX – 5-FU : Encapsulation and Release

EE(%) = (Dt−Df / Dt)×100

Dt = total theoretical amount of drug added (mg/mL)Df = concentration of free drug after encapsulation (mg/mL)

Release kinetic mathematical models

Zero-order

First order

Higuchi

Hixson-Crowell

Kosmeyer-Peppas

tKQQ t 00

tKQQt 10loglog

5.0

0 tKQQ Ht

tKQQ HCt 30

n

KPt tKQ

UV-Vis spectroscopy

DOX 480 nm TMZ 328 nm 5-FU 525 nm

Results

Results : CS-g-PLACA nanoparticles

Influence of polymer / DS ratio (w/w) on nanoparticles size and z-potential pH 5.5 DLS analysis

-40

-30

-20

-10

0

10

20

30

40

50

60

0 2 4 6z-p

ot.

(mV

)polymer / DS ratio (w/w)

Dependence of nanoparticles dimension on polymer/DS ratio (w/w) Best compromise between size and z-potential is presented at w/w ratio 2 Change in polymers chains arrangement

0

50

100

150

200

250

0.1 0.5 1 2 5

polymer / DS ratio (w/w)

CS CS-g-PLAСA

Siz

e(n

m)

Results : SEM and TEM analysis

CS-g-PLACA Dextran Sulfate

+ -

+

+

+

+

+

+

+

--

--

-

-

Results : CS-g-PLA nanoparticles

0

50

100

150

200

250

300

0 50 100 150 200 250

Siz

e (n

m)

Time (h)

pH 5.5 pH 7.4

CS

0

50

100

150

200

250

300

0 50 100 150 200 250

Siz

e (n

m)

Time (h)

pH 5.5 pH 7.4

CS-g-PLACA

Stability studies : nanoparticles dimension VS time Influence of pH T : 37 °C Polymer / DS (w/w) : 2

PLACA side chain increases nanoparticles stability

I

II II

I

Results: Single Loading

0

20

40

60

80

100

3.5 5.5 7.4 9En

cap

sula

tio

n E

ffic

ien

cy

(%)

pH

DOX

TMZ

5-FU

0

20

40

60

80

100

3.5 5.5 7.4 9

En

cap

sula

tio

n e

ffic

ien

cy

(%)

pH

DOX

TMZ

5-FU

CS CS-g-PLACA

Room temperature Polymer / DS (w/w) : 2

pH 5.5 represents the optimal condition Drugs are well balanced in both systems CS-g-PLACA shows higher encapsulation efficiency than CS

CS CS-g-PLACA5%

0

20

40

60

80

100

3.5 5.5 7.4 9pH

TMZ DOX

0

20

40

60

80

100

3.5 5.5 7.4 9pH

TMZ DOX

0

20

40

60

80

100

3.5 5.5 7.4 9pH

5-FU DOX

0

20

40

60

80

100

3.5 5.5 7.4 9pH

5-FU TMZ

0

20

40

60

80

100

3.5 5.5 7.4 9pH

5-FU DOX

0

20

40

60

80

100

3.5 5.5 7.4 9pH

5-FU TMZ

Co

-En

cap

sula

tio

n E

ffic

ien

cy (

%)

Co

-En

cap

sula

tio

n E

ffic

ien

cy (

%)

Results: Multiple Loading

Results: CS-g-PLACA- Release kinetics

0

20

40

60

80

100

0 100 200 300 400 500

Cu

mu

lati

ve

rele

ase

(%)

Time (h)

DOX TMZ 5-FU

0

10

20

30

40

50

60

70

0 10 20 30

Cu

mu

lati

ve

rele

ase

(%)

Time (h)

DOX, TMZ and 5-FU loaded separately Physiological solution (pH 7.4) T : 37 °C

No initial burst effect is observed in all formulations Release begin after 5 hours

tlag

Common trend

Results: CS-g-PLACA-Release kinetics

DOX, TMZ and 5-FU loaded simultaneously Physiological solution (pH 7.4) T : 37° C

0

20

40

60

80

100

0 100 200 300 400

Cu

mu

lati

ve

co-r

elea

se (

%)

Time (h)

DOX TMZ 5-FU

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30

Cu

mu

lati

ve

co-r

elea

se (

%)

Time (h)

Common trend

DOX, TMZ and 5-FU are released concurrently No initial burst effect is observed in all formulations Release begin after 4 hours

tlag

Release Kinetic mathematical analysis

Zero-order(R2)

First-order(R2)

Higuchi (R2)

Hixson-Crowell(R2)

Korsmeyer-Peppas(R2)

TMZ 0.73 0.83 0.97 0.97 0.90

DOX 0.88 0.85 0.98 0.98 0.91

5-FU 0.79 0.81 0.98 0.91 0.92

Physiological solution pH 7.4 T : 37° C CS-g-PLACA

Higuchi model shows the best fit Drugs are released by diffusion R2 values in Korsmeyer-Peppas models indicates that the Fickian diffusion

represents the controlling factor Results related to Hixson-Crowell model demonstrate that a slight change

in the nanoparticles surface area is induced by the media

Conclusions

CS-g-PLACA based nanoparticles show dimension in the range 100-200 nm

Nanoparticles show good stability as far as 10 days

Up to 70% of encapsulation efficiency of TMZ, 5-FU and DOX

No initial burst effect at physiological condition

Balanced release of drugs when loaded simultaneously

Outlooks

Cell uptakes studies

Evaluation of citotoxicity in different cancer cells lines

In vivo tests

Results : PLACA

UV-Vis

FTIR-ATR

1H-NMR

300 MHzConc : 1% (w/w) in DMSO-d690 pulse angle

Zn-Se crystalResolution : 2cm-1

Conc: 0.1 mg/mL in CHCl3Resolution : 0.5 nmPath 10mm

Results: CS-g-PLACA

FTIR-ATR

1H-NMR