Human neuropeptide Y (NPY) is a neurotransmitter and hormonal peptide that is undergoing intensive investigation for the treatment of cachexia, certain psychiatric disorders and neuropathic pain.1, 3, 4, 39, 40
Delivery of NPY directly to peripheral tissues possess an additional interest and can be beneficial when peptide action in central nervous system (CNS) is not required. Anti-proliferative and proapoptotic10
effects of NPY mediated through Y(1)
receptor activation can be exploited in treatment of solid tumors overexpressing this receptor subtype. Moreover, targeting of peripheral energy utilization6
as supplementary treatment of anorexia and downregulation of T-helper 1 hypersensitivity41
in acute stages of inflammation with NPY are therapeutically beneficial. Besides direct therapeutic application, labeled NPY analogs could serve as a diagnostic tool in imaging. This approach was recently tested in humans to image breast cancer and metastasis with technetium 99 tagged NPY.42
We anticipate that labeled NPY in SSM should be more effective in imaging due to enhanced accumulation at a tumor site through passive and active targeting.
However, fast proteolytic degradation of NPY upon administration is a major barrier for its successful clinical application.2
The new findings of this study showed that NPY, which underwent spontaneous self-association with SSM, was protected from enzymatic degradation and its bioactivity was significantly amplified.
Self-association of monomeric NPY with SSM abrogated formation of peptide aggregates in aqueous environment (saline) as shown by particle size analysis (). We postulate that intermolecular hydrophobic interactions between NPY molecules at the dimer/tetramer interface are superseded by more energetically-favorable interactions of NPY molecules with PEGylated phospholipid micelles. This was further evidenced by increased fluorescence emission of NPY in the presence of SSM (). The enhanced fluorescence intensity was likely due to reduced fluorescence quenching between aggregated NPY molecules.21
To this end, we found that up to three NPY molecules self-associate with each micelle freely, without interaction with each other.
Furthermore, our circular dichroism study showed that the association of NPY with biocompatible and biodegradable sterically stabilized phospholipid micelles in aqueous environment resulted in stabilization of α-helical peptide confirmation (). This was in agreement with the results of another research group on NPY conformational changes with a model membrane system (dodecylphosphocholine micelle). This study revealed a raise in NPY α-helicity, particularly in its C-terminal segment upon interaction with the dodecylphosphocholine micelles.11
Given that the C-terminal of NPY is critical for binding to its targets, this conformational change favors peptide-receptor interaction.11, 20, 43, 44
Increased α-helicity of NPY in SSM is likely contributed, in part, by the adoption of helical conformation at the C-terminal pentapeptide. Moreover, NPY most likely reside within the PEG palisade of PEGylated micellar system, rather than binding to the phospholipid headgroup surface as claimed with dodecylphosphocholine micelles free of PEG corona.11
Therefore, the PEG layer of SSM should provide an additional steric barrier against physical aggregation and enzymatic degradation of NPY, which would result in further increase in the stability of NPY.
This notion is further supported by our findings that self-association of NPY with SSM abrogated enzymatic peptide cleavage induced by DPP-4 () and potentiated NPY bioactivity as indicated by enhanced suppression of cAMP accumulation in forskolin stimulated SK-N-MC cells (). These cells express neuropeptide Y(1)
receptors that upon activation reduce intracellular cAMP accumulation which underlies many of the NPY-induced central and peripheral responses.6, 11–13, 15, 37
Since SK-N-MC cells have been reported to exogenously express DPP-4,45
enhanced bioactivity of NPY-SSM in these cells could be related, in part, to NPY molecule protection within SSM. However, we believe that such protection is “reversible” and do not interfere with receptor/ligand interaction. This, in turn, enables more intact full length NPY molecules in favorable α-helix conformation to interact with their receptors thereby amplifying peptide bioactivity. Moreover, the peptide will be presumably protected by SSM from the enzymatic cleavage not only in vicinity of their cell targets but also in circulation prolonging its half-life. The possibility of interaction of NPY in SSM with Y(2)
receptors can not be ruled out. Clearly, additional studies are needed to evaluate this fact.
The results of this study represent the first step in our overall goal to safely and effectively deliver human NPY by the intravenous or intranasal routes. The former path is more applicable for peripheral peptide delivery since our nanomicellar technology most likely will not allow blood brain barrier crossing. From another hand, feasibility of intranasal delivery of peptide drugs has been shown for various nanoparticulate carriers, including liposomes and PEGylated polymeric nanoparticles46–48
and should be applicable for NPY-SSM as well. Moreover, NPY is currently being tested by intranasal route on humans in a new clinical trial,49
but not as a nanoparticulate formulation.
In conclusion, we found that self-association of NPY with sterically stabilized phospholipid micelles amplifies its bioactivity in vitro
. This may be related, in part, to decreased aggregation and increased stability of NPY molecules by the carrier. This, in turn, obviates the need to chemically modify NPY molecule to improve its stability thereby circumventing possible immunogenicity and adverse events.24, 28, 50
Phospholipid micelles used in this study are composed of PEGylated lipid, which is a component of U.S. FDA approved product Doxil®. The final product, NPY-SSM dispersion is easy to prepare, scale up and freeze-dry for long-term storage. Therefore, we propose further development of NPY in SSM as a novel, long-acting nanomedicine for conditions with peptide deficiency and where NPY/receptor Y(1) pathway mediation is needed.