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Novel PLA-Chitosan nanocomplexes for controlled and sustained release of DNA-Alkylating drugsA. Di Martino, A. Pavelkova, P. Kucharczyk, V. Sedlarik

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

dimartino@ft.utb.cz

approaches, formulations, technologies for the targeted delivery and/or controlled release of therapeutic agentsSafePerform therapeutic functionConvenient administrationSimple to manufactureDrug 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) 38

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Development of DDS : an overview

Park. Journal of Controlled Release 190 (2014) 38

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 Various sources : Algae, Microbial, Plants and animals

Abundant

Wide range of Mw

Chemical composition

Large number of reactive groups

Biocompatible and biodegradable

Low immunogenicity

Not toxic

Why polysaccharides?

Polysaccharides in Drug delivery

Hyaluronic acidAlginate

Pectic acid

Dextran sulfate

Cyclodextrins

Chondroitin sulfate

Chitosan

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Small molecules Proteins PeptidesCyclodextrinesPolysaccharidesDrugsChitosan and derivativesSynthetic PolymersLMW, MMW,HMW

-C3-OH-C6- OH-NH2D-GlucosamineN-Acetyl -D-Glucosamine

GoalsPreparation of a set of amphiphilic carriers

Single and Multiple Encapsulation of DNA alkylating drugs

Improve drug release performance

Reduction of burst effect

Increase drug cytotoxicity

Polylactic acid (PLA)Linear PLA (LPLA)Carboxy enriched PLA (CPLA)Branched PLA (BPLA)Amphiphilic polymerChitosan

Low molecular weight

D.D. 75-85 %Hydrophilic backbone

Hydrophobic side chain

Chitosan-PLA synthesis and characterization

FTIR-ATR

1H-NMRSynthesisCharacterizationDi Martino et al. Int.J.Pharm. 2015 Dec 30;496(2):912-21Di Martino & Sedlarik . Int.J.Pharm. 2014 Oct 20;474(1-2):134-45

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

PECPolyions- Natural- Semi synthetic- Synthetic

+ charges / - chargesMwConcentration

IGSmall molecules

Nanoparticles dimension Core structure Stability Encapsulation efficiency Release ratePEC or IGWhat are we looking for ?

+ charges / - chargesConcentration

Drug LoadingPost preparation

Low encapsulation efficiency

Surface localization

Burst effect Preparation

High encapsulation

Protection from external environment

Sustained release

Burst effect reductionParameters Carrier structure Drug structure Method pH Temperature Ionic strength Drug to carrier weight ratio

Carrier Drug interactions

Anticancer drugs

Suicide inhibitor Inhibition of thymidylate synthase AntimetaboliteDoxorubicinTemozolomide5-Fluorouracil Alkylate/methylate DNA N-7 or O6 guanine Resistance mechanism DNA intercalation Inhibits topoisomerase II Block the DNA replicationSIDE EFFECTSHYDROLYSIS CIRCULATION TIME

SIDE EFFECTSADMINISTRATION ROUTE

Why use nanocarriers ?

Nanocomplexes 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 loadingCarrier structure-Drug structure-Environment conditions

Environment Presence of side chain (-COOH, branched) Drug structureInfluence encapsulation efficiency

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

Encapsulation efficiency-multiple loading

DOX containing formulations are better encapsulatedFurther investigations

Release kinetic models Statistical methods Model dependent methods Model independent methods exploratory data analysis method repeated measures design multivariate approach Zero & first order, Higuchi model, Korsmeyer-Peppas model, Hixson Crowell, Baker-Lonsdale model, Weibull model .difference factor (f1) and similarity factor (f2) to compare dissolution profiles

Reduction of burst effectLarge amount of drug released immediately upon placement in the mediaAdvantagesWound treatmentTargeted delivery (triggered burst release)Pulsatile release

DisadvantagesLocal or systemic toxicityIn vivo short t1/2Waste of drugShort release profileFrequent administrationDifficult to predict intensity

Journal of Controlled Release 73 (2001) 121 136What is burst effect?

Release rate - Individual

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

No initial burst effect is observed in all formulationsRelease begin after 5 hoursSustained release

tlag

Release rate - Simultaneous

No interactions among drugsNo initial burst effect is observed in all formulationsRelease begin after 4 hoursSustained release

tlag

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

Cell tests Cytotoxicity evaluation of free and loaded drugs

MEF, NIH/3T3 cell-linesCS-BPLACS-BPLA+ 5FUCS-BPLA + TMZCS BPLA + DOX

CS-LPLACS-BPLA

Presence of PLA side chain increase drug cytotoxicity!!!24h24h24h24h

Conclusions Nanocomplexes 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 nanocomplex 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

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

Acknowledgements

grant No. 15-08287Ygrant No. LO1504