Diapozitiv 1

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Formulation of PLGA nanoparticles for
intracellular delivery of protein drug
Dr. Mateja Cegnar, M. Pharm.
Faculty of Pharmacy, University of Ljubljana,
Slovenia
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OUTLINE

• Model protein drug cystatin
• Pharmaceutical formulation for the protein drug
• Why nano-sized system?
• Production of polymer nanoparticles with
  cystatin
• Mechanism of NPs formation
• Effect of process parameter on NP-size and
  cystatin activity
• Cellular assays using MCF-10A neoT cell line
• Delivery of cystatin into the tumour cells by NPs
• Efficiency of cystatin delivered intracellular by
  NPs

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Model protein drug - cystatin

• Isolated from chicken egg white
• Contains 115 amino acid residues (13 kDa)
• Constant of inhibition to cathepsin B (10-10M)
• Extracellular inhibitor of cysteine proteases

?
Intracellular delivery ? suitable carrier system
?
Incorporation in NANOPARTICLES
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Pharmaceutical formulation of the protein drug
? Why ?
To increase the bioavailability of protein drug
and to prolong its short circulation life time in
biological environment (pH, enzymes, lipid
membranes )
? How ?
Physical entrapment of protein into colloidal
carriers (10-1000 nm) nanosized systems
(protection by the carrier matrix, modified
release, site selective delivery ...)
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Why nano-scale carrier for cancer ?
Size ? affects the biodistribution profile and
therapeutical bechaviour of the system
Nano-size ? penetration, cellular uptake,
targeting
?

• active
• passive

Normal tissue
Tumour tissue
Intratumoural delivery of NPs
Defective lymphatic drainage
Dis-organized and leaky tumour endotelium
EPR-effect Enhanced permeation and retention
effect
Normal vessels with tight endothelium
Lymph node
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Production of cystatin-loaded NPs
Materials

• Cystatin labelled with a fluorescent dye Alexa
  Fluor 488

• Carrier material copolymer of lactic and
  glycolic acid

• Organic solvent ethyl acetate (partial
  solubility in water)

• Stabilizer polyvinyl alcohol

Method
A double emulsion solvent diffusion method
Technology
Low energy emulsification using the combination
of mechanical stirring and bath sonication
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Mechanism of nanoparticle formation emulsion
method
emulsion system
nanoparticle dispersion
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Production of cystatin-loaded PLGA NPs
4. Hardening of nanoparticles
2. Formation of first emulsion
3. Formation of double emulsion
1. Addition of protein phase to polymer solution
large volume of aqueous phase
emulsifier PVA (aq)
energy
energy con.
energy

• polymer in organic solvent
• protein in aqueous phase

7. Lyophilization of NPs
5. Washing of NPs
6. Filtration of NPs
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Effect of technological parameters on NP-size and
cystatin activity
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SEM image of PLGA nanoparticles with cystatin
Characterization Size 300-350 nm Cystatin
loading 2.1, EE 45 Release 70 burst release
Supra 32VP instrument (Oberkochen, Zeiss,
Germany)
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Cellular assays using MCF-10A neoT cell line
1. Delivery of cystatin into the cells by NPs
2. Efficiency of cystatin delivered
intracellular by NPs

• identification and localization of
  the intracellular target cathepsin B
• inhibition of intracellular proteolysis with
  delivered cystatin

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Internalization of NPs into MCF-10A neoT cells
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Identification of intracellular cathepsin B
(localisation/activity)
Proteolytic activity of cathepsin B using the
fluorogenic substrate Z-Arg2 cresyl violet
Immunocytochemical identification of cathepsin B
using specific 3E1 monoclonal antibody
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Inhibition of intracellular proteolysis
Nanoparticle-delivered cystatin
Free cystatin
Cathepsin B proteolysis
Inhibition of cathepsin B proteolysis
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Summary

• Successful formulation of PLGA nanoparticles
  (300-350 nm) containing active cystatin (85)
• Time dependant cellular uptake of NPs
• Facilitated intracellular delivery of cystatin by
  NPs
• Inhibition of intracellular proteolysis by
  nanoparticle-delivered cystatin

Conclusion

• Suitable pharmaceutical formulation can increase
  the biological effectiveness of protein drugs

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Acknowledgments
Faculty of Pharmacy University of Ljubljana
Academic Medical Centre Amsterdam
Cornelius J. Van Noorden
Julijana Kristl Janko Kos
Joef Stefan Institute Department of Biochemistry
and Molecular Biology
Valentina Zavanik-Bergant Ale Premzl