Novel amphiphilic nano-carrier based on PLA-polysaccharide for multi-drug encapsulation and controlled release.

In Vitro evaluationA. Di Martino, P. Kucharczyk, Z. Kucekova, P. Humpolicek, V.Sedlarik

Centre of Polymer Systems, University Institute, Tomas Bata University in Zlín, tr. T. Bati 5678, 76001 Zlin, Czech Republic

«approaches, formulations, technologies for the targeted delivery and/or controlled release of therapeutic agents»

Safe Perform therapeutic function Convenient administration Simple to manufacture

Drug Delivery Systems (DDS)

1950 – 1980 : 1st generation – Basics of controlled release

1980 – 2010 : 2nd generation – Smart delivery

2010 – 2040 : 3rd generation – Modulated delivery

- Zero order- Smart polymers and hydrogels- Peptide and protein- Nanoparticles

- Targeted delivery- Long term delivery systems - Minimal burst- In vitro-in vivo correlation (IVIVC)

Park. Journal of Controlled Release 190 (2014) 3–8

Nanoparticles as DDSWhy use Nanoparticles?

Dispersion or solid form Various morphologies – nanospheres, nanocapsules……. Drug(s) can be dissolved, entrapped, encapsulated or attached High Encapsulation and Loading Efficiency Protection Controlled and sustained release Side effects reduction

Polysaccharides in Drug delivery

Hyaluronic acidAlginate

Pectic acid

Dextran sulfate Cyclodextrins

Chondroitin sulfate

Chitosan

5

Small molecules

Proteins Peptides

CyclodextrinesPolysaccharides Drugs

Chitosan and derivatives

Synthetic PolymersLMW, MMW,HMW

-C3-OH-C6- OH

-NH2

D-Glucosamine N-Acetyl -D-Glucosamine

Polylactic acid (PLA)

Biomedical application of PLA

LPLA – Low molecular weight linear PLACPLA – Low molecular weight Carboxy enriched PLABPLA – Low molecular weight Branched PLA

GoalsPreparation of a set of amphiphilic carriers

Single and Multiple Encapsulation of anticancer drugs

Improve drug release performance and stability

Reduction of burst effect

Increase drug cytotoxicity

Chitosan-g-PLA : synthesis and characterization

Synthesis Coupling reaction between CS-NH2 and PLA-COOH EDC/NHS Low conjugation degree Further conjugation (Folic acid, Fluorescein, DTPA..) CS properties conserved

Characterization : FTIR-ATR 1H-NMR

Di Martino et al. Int.J.Pharm. 2015 Dec 30;496(2):912-21

CS-PLA based nanoparticles Polyelectrolytes Complexation (PEC)

Ionotropic Gelation (IG)

Fast and simple Solvent free Not needs special equipment Dimension and surface charge control Low P.D.I. Good reproducibility

PEC Dextran sulfate • + charges / - charges•Mw•Concentration

IG• + charges / - charges•Concentration

Characterization : DLS, SEM, TEM

Anticancer drugs

Suicide inhibitor Inhibition of thymidylate synthase Antimetabolite

Doxorubicin Temozolomide 5-Fluorouracil

Alkylate/methylate DNA N-7 or O6 guanine Resistance mechanism

DNA intercalation Inhibits topoisomerase II Block the DNA replication

SIDE EFFECTSHYDROLYSIS

CIRCULATION TIME SIDE EFFECTSADMINISTRATION ROUTE

Nanoparticles characterization

- Single loading not influence average dimension - Simultaneous loading increase average dimension

PLA side chain structure direct influences nanoparticles size

Increase in dimension up to 50 %

Average dimension is around 150nm

Encapsulation Efficiency- single loading

Environment Presence of side chain (-COOH, branched) Drug structure

Influence encapsulation efficiency

Di Martino & Sedlarik . Int.J.Pharm. 2014 Oct 20;474(1-2):134-45

Encapsulation efficiency-multiple loading

DOX conteining formulations are better encapsulated Further investigationsDi Martino & Sedlarik . Int.J.Pharm. 2014 Oct 20;474(1-2):134-45

Bust Effect Reduction

Large amount of drug released immediately upon placement in the media

Advantages Wound treatment Targeted delivery (triggered burst release) Pulsatile release

Disadvantages

Local or systemic toxicity In vivo short t1/2 Waste of drug Short release profile Frequent administration Difficult to predict intensity

Journal of Controlled Release 73 (2001) 121 –136

What is burst effect?

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 1 2 3 4 5 60

10

20

30

40

50

60

DOX

TMZ

CS

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 1 2 3 4 5 60

10

20

30

40

50

60DOX

TMZ

CS-g-PLA

40%

30%

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 1 2 3 4 5 60

10

20

30

40DOX

TMZ

CS-g-PLACA2%

lag time

20%

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 1 2 3 4 5 60

10

20

30

40DOX

TMZ

CS-g-PLACA5%

lag time10%

Sustained-release Delayed-release

Release kinetic : Burst Effect reduction and Delayed Release

Improve drug stability

N

N

N

NN

ONH2

CH3O

N

NH

O NH2

NN NH

CH3N

NH

NH2

O NH2

N+

NCH3

+TMZ MTIC

AIC

Diazomethane cation

H2O

-CO2

TMZ quickly hydrolyse in physiological condition

Improve stability of TMZ is a challenge

TMZ free in PSTMZ loaded in CS-g-BPLA / CPLA

t1/2 : 35 – 180 min

t1/2 : few minX100 120 140 160 180 200 220 240

0

1

2

3

4

5

6

7

8

9

10

m/z

Cou

nts

x105

TMZPS 6h

100 120 140 160 180 200 220 2400

1

2

3

4

5

6

7

8

9

10

m/z

Cou

nts

x105

TMZPM 6h

TMZ

TMZ

MTIC

AIC

3h

6h

Cell tests - Citotoxicity Citotoxicity evaluation of free and loaded drugs

MEF, NIH/3T3 cell-lines

CS-BPLA CS-BPLA+ 5FUCS-BPLA + TMZCS –BPLA + DOX

CS-LPLACS-BPLA

Presence of PLA side chain increase drug citotoxicity!!!

24h24h 24h 24h

Conclusions Nanoparticless based on chitosan and a set of different PLA were prepared

Dimension up to 200 nm and good stability in various simulated physiological fluid

PLA side chain directs influence the nanoparticles properties

Presence of COOH groups and branched PLA structure increase encapsulation efficiency

Reduction of burst effect BPLA > CPLA > LPLA

Branched PLA prolongs the release of all tested drugs

Prepared CS-PLA based nanoparticles delay TMZ hydrolysis in physiological condition

Cell citotoxicity tests demonstrate an increase in drug toxicity after loading in CS-PLA based formulations

Acknowledgements

grant No. 15-08287Y

grant No. LO1504