chitosan grafted carboxy functionalized polylactide nanoparticles for multidrugs controlled and...
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Chitosan grafted carboxy functionalized polylactidenanoparticles for multidrugs controlled and sustained
release
Antonio Di Martino
Center of Polymer SystemsTomas Bata University in Zlin
Czech Republic
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