Release and bioactivity of PACA nanoparticles containing D-Lys6-GnRH for brushtail possum fertility control
Graphical Abstract
Introduction
The common brushtail possum (Trichosurus vulpecula) is a marsupial herbivore native to Australia that has become a pest species in New Zealand. The brushtail possum causes significant damage to native flora and fauna and is a reservoir for Mycobacterium bovis, the bacterium causing bovine tuberculosis [1]. Current methods of control (poisoning, shooting and trapping) have the disadvantage that they lack target specificity and humaneness, and they have failed to reduce the population of brushtail possums long term. The most publicly accepted strategy of possum population control is reducing their fertility [2]. Fertility control agents are likely to be protein and peptide bioactives [3], such as the neurohormone gonadotropin releasing hormone (GnRH), a key compound in the regulation of reproduction. D-Lys6-GnRH is a potent GnRH agonist and may be employed as a regulatory hormone causing a downregulation of GnRH receptors and desensitization of pituitary gonadotroph cells. Alternatively, GnRH may be used as a toxin conjugate, such as the GnRH-PAP (pokeweed antiviral protein) conjugate, which is able to target gonadotropin producing cells specifically and cause cell death [1], [4].
Any form of biocontrol requiring the individual handling of the animal is not feasible for wildlife. Self disseminating delivery systems (e.g. genetically modified viruses) raise safety concerns considering the proximity of New Zealand to Australia's protected marsupials [5]. In contrast, nondisseminating systems are safer but require aided delivery via bait and include carriers such as bacterial ghosts [6], [7], virus like particles (VLPs) [1] and pharmaceutical polymeric nanocarriers [3]. Bacterial ghosts and VLPs largely retain their immunogenicity and so appeal as a delivery system for immunocontraceptives. Chemical sterilitants, such as GnRH derivatives, however, are direct acting and should avoid the immune system. Pharmaceutical nanocarriers, such as poly(alkylcyanoacrylate) (PACA) nanoparticles can be prepared not only to avoid the immune system but also to protect the bioactive and facilitate the uptake in the gut, while maintaining the bioactivity of the drug [8], [9], [10], [11]. Furthermore, PACA nanoparticle formulations are easy to scale up, reproducible and affordable [12].
In situ loading of protein and peptide bioactives into PACA nanoparticles prepared by interfacial polymerization results in nanocapsules that confine the bioactive to the aqueous core and the polymer wall [13], [14]. High entrapment efficiencies are reported for proteins and peptides [13], [15], [16]; however, they are accompanied by low release rates of drug in the absence of esterases [17], [18]. Previously, we have demonstrated the covalent interference of some protein and peptide drugs, including D-Lys6-GnRH, during the polymerization process, which may explain the low release of bioactive observed in some studies [19], [20]. The sustained release of bioactive via the bioerosion of the polymer material has previously been suggested for peptide copolymerized PACA nanoparticles in the presence of esterases and rat plasma [21]. Whether or not peptide released from copolymerized PACA nanoparticles is still bioactive has been investigated only in a few studies. Gibaud et al. [17] demonstrated that the bioactivity of recombinant human granulocyte colony stimulating factor (rhG-CSF) in situ polymerized with PACA nanoparticles was lost. However, some authors have suggested that, despite the covalent association of proteins and peptides with PACA polymer, the in vivo bioactivity was maintained [18], [22]. Tasset et al. [18] demonstrated the hypocalcemic effect of i.v. administered calcitonin-loaded PACA nanoparticles was equivalent to the an i.v. dose of free drug. Peptide copolymerized systems may maintain their bioactivity if the bioactive site remains unobstructed and the folding of the bioactive is unaffected by the modification. For D-Lys6-GnRH, we have demonstrated that the modification occurs at the histidine residue [20], which is involved in receptor binding [23].
The aim of this study was to investigate the potential of PACA nanoparticles to deliver the fertility control agent D-Lys6-GnRH to a wild target species, the brushtail possum, and induce a biological response. Poly(ethylcyanoacrylate) (PECA) nanoparticles containing D-Lys6-GnRH were characterized in vitro for the amount of peptide released under physiological conditions. The in vivo bioactivity, measured as the plasma concentration of luteinizing hormone, was also determined following intracaecal administration in the brushtail possum.
Section snippets
Materials
D-Lys6-GnRH (p-Glu-His-Trp-Ser-Tyr-Lys6-Leu-Arg-Pro-GlyNH2) was purchased from PolyPeptide Laboratories (Torrance, CA, USA). Ethyloleate GPR was sourced from BDH Laboratory Supplies (Poole, England) and surfactants sorbitan monolaurate (Crill 1™) and ethoxy 20 sorbitan mono-oleate (Crillet 4™) were kindly provided by BTB Chemicals (Auckland, New Zealand). The monomer ethylcyanoacrylate (ECA) (Sicomet 40) was kindly donated by Henkel Loctite (Hannover, Germany). Sodium chloride 0.9%,
Physicochemical characterization of PECA nanoparticles containing D-Lys6-GnRH
PECA nanoparticles in situ loaded with D-Lys6-GnRH were on average 190 ± 0.5 nm in size with a polydispersity index of 0.077 ± 0.005 and a zeta potential of + 8.78 ± 0.183 mV. This is in consensus with our previous work [20]. The positive zeta potential gives a clear indication of the contribution of the positively charged peptide to the overall net charge of the otherwise negatively charged PECA nanoparticles either via the adsorption or copolymerization of peptide. The entrapment efficiency for D-Lys6
Conclusions
PECA nanoparticles comprise a feasible formulation strategy for the peroral delivery of the chemical sterilitant D-Lys6-GnRH to a target species, the brushtail possum. PECA nanoparticles in situ loaded with D-Lys6-GnRH showed little release (< 5%) of bioactive in AGJ and SIF, while the particles stayed intact over the duration of 6 h (TEM) and retained large fractions of intact peptide (MALDI TOF MS). In contrast, about 60% of D-Lys6-GnRH was released within 1 h when incubated in the presence of
Acknowledgements
The study was funded by FRST (Foundation for Research, Science and Technology), New Zealand. Acknowledgement is given to AgResearch Invermay New Zealand for their help with the animal experiments (Euan Thompson and Taryn Skinner) and analysis of blood samples (Shirley Martin), the Microscopy Unit (Department of Anatomy and Physiology, University of Otago) for their support with TEM imaging and Dr. Torsten Kleffmann (Centre for Protein Research, Department of Biochemistry, University of Otago)
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Present address: Coriolis Pharma, Am Klopferspitz 19, 82152 Martinsried, Germany.