Histidine residues in the peptide D-Lys(6)-GnRH: potential for copolymerization in polymeric nanoparticles.
Poly(ethylcyanoacrylate) (PECA) nanoparticles containing the bioactive d-Lys(6)-GnRH were manufactured by an in situ interfacial polymerization process using a w/o-microemulsion template containing the peptide in the dispersed aqueous pseudophase of the microemulsion. Polymeric nanoparticles were characterized using PCS, RP-HPLC (bulk level) and MALDI TOF mass spectrometry (molecular level). The peptide d-Lys(6)-GnRH was reactive with the alkylcyanoacrylate monomer, resulting in some of the peptide copolymerizing with the monomer. MALDI TOF/TOF (tandem) analysis revealed that the histidine residue in position 2 of d-Lys(6)-GnRH interacts covalently in the polymerization process. A reaction mechanism for this nucleophilic interference is suggested. The copolymerization reaction appeared to occur within seconds after the addition of the monomer to the microemulsion. The surface charge of resulting nanoparticles was less negative (-3 mV) compared with the zeta potential of empty nanoparticles (-27.5 mV). The copolymerization yielded high entrapment rates of 95 +/- 4% of peptide, but showed limited release ( approximately 11%) of free peptide over 5 days. A separate experiment demonstrated that the addition of d-Lys(6)-GnRH to preformed empty PECA nanoparticles (ex situ) also yielded fractions of copolymerized peptide suggesting a certain proportion of polymer remains available for copolymerization possibly through an unzipping depolymerization/repolymerization process. Therefore, the reactivity of histidine residues in bioactives needs to be considered whenever using the bioactive in situ or ex situ with polymeric PECA nanoparticles.
Kafka
Alexandra P
AP
School of Pharmacy, University of Otago, Dunedin 9054, New Zealand.
Kleffmann
Torsten
T
Rades
Thomas
T
McDowell
Arlene
A
D000327:Adsorption; D003487:Cyanoacrylates / Q000737:chemistry; D004337:Drug Carriers; D016503:Drug Delivery Systems; D007987:Gonadotropin-Releasing Hormone / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D053758:Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology; D010316:Particle Size; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D003487:Cyanoacrylates; D004337:Drug Carriers; D007987:Gonadotropin-Releasing Hormone; D006639:Histidine; C035029:LHRH, Lys(6)-