8thAItUN Annual Meeting
Medicines for Children’s safe:
challenges and opportunities
Pavia, Italy●6-7 March 2014
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Peptide-functionalized PLGA nanocarriers as a platform for EGFR overexpressing cells targeting
a,*Colzani B., aDorati R., aConti B., bBiagiotti M., bSperanza G., and a,*Genta I.
aUniversity of Pavia, Dept. Drug Sciences, V.le Taramelli,12 – 27100 Pavia (Italy)
bUniversity of Milan, Dept. Chemistry, Via Golgi, 19 – 20133 Milano (Italy)
*
Purpose
To exploit the agent YHWYGYTPQNVI (GE11), a dodecapeptide that has been demonstrated to selectively recognise the epidermal growth factor receptor (EGFR), in order to realize selective nanosized drug delivery systems by combining passive and active cell targeting.
To set-up chemical and technological protocols to obtain a GE11-poly-lactide-co-glicolide conjugate (GE11-PLGA) as “smart” nanoparticulate platform for drug delivery.
Methods
A model tetrapeptide (FQPV, Mw 489.57 g/mol) and GE11 were synthesized by standard fluorenyl-9-methoxy-carbonyl (Fmoc) protocol using a Biotage Initiator SP Wave synthesizer. They were purified by semi-preparative HPLC using an AKTA Basic100 instrument and its purity characterized by analytical RP-HPLC. The peptides identity and molecular weight were confirmed by MALDI TOF mass spectrometry (Bruker Microflex LT Spectrometer).
FQPV was used to set up the best protocol for chemical conjugation to PLGA (7525 DLG 3A, Mw 35,000 Da, Lakeshore Biomaterials, USA) (FQPV-PLGA), using carbodiimmide chemistry. FQPV-PLGA and GE11-PLGA were characterized by nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC).The FQPV peptide was labelled with fluorescein in order to quantify, by fluorescence spectrometry, the amount of peptide covalently bound to the polymer.
FQPV-PLGA and fluorescein-labelled-FQPV-PLGA nanoparticles were prepared by suitably set up nanoprecipitation technique. Fluorescein-labelled-FQPV loaded PLGA nanoparticles were also prepared by adsorption or encapsulation protocols in order to compare different techniques to load peptide to the polymer.
The nanoparticles were characterized for their morphology (TEM), particle size and Z-potential (NICOMP 380 ZLS apparatus), differential scanning calorimetry (DSC) and peptide loading (fluorescence spectrometry).
Results
GE11 peptide was obtained with a 35% yield and its purity was shown to be > 95%. NMR spectra showed covalent bond of FQPV peptide to PLGA, as further demonstrated by fluorescence spectrometry.
Placebo PLGA nanoparticles showed homogeneous size distribution and suitable dimensions (330.0±77.200nm, PI 0.055). Zeta potential was -4.27 mV. FQPV-PLGA nanoparticles didn’t show significant differences in terms of dimensions and surface charge. TEM images confirmed nanoparticle size distribution and revealed regular spherical shape.
Conclusions
The developed synthetic procedure represents a good platform for PLGA functionalization with different peptides, aimed at preparing smart nanoparticles. This work is a preliminary part of a major project which goal is the preparation of nanoparticles selective to EGFR overexpressing cells.