Journal
BIOMOLECULES
Volume 12, Issue 10, Pages -Publisher
MDPI
DOI: 10.3390/biom12101362
Keywords
peptides; relaxin; lipidic conjugate; liposomes; stability; biological activity
Categories
Funding
- Western Greece Region [MIS 5021443]
- programme MEDICUS of the University of Patras -Research Committee [81762]
- Public Investment Programme of Greece [MIS 5021443]
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The encapsulation of RLX2 in liposomes and the synthetic modification of P-RLX2 can both prolong the stability of RLX2 peptide in vitro. However, the chemical conjugation leads to a significant loss of bioactivity, while the effect of liposome entrapment on RLX2 activity is less pronounced.
Relaxin (RLX) is a protein that is structurally similar to insulin and has interesting biological activities. As with all proteins, preservation of RLX's structural integrity/biological functionality is problematic. Herein, we investigated two methods for increasing the duration of relaxin-2's (RLX2) biological activity: synthesis of a palmitoyl RLX2 conjugate (P-RLX2) with the use of a Palmitoyl-l-Glu-OtBu peptide modifier, and encapsulation into liposomes of P-RLX2, RLX2, and its oxidized form (O-RLX2). For liposomal encapsulation thin-film hydration and DRV methods were applied, and different lipid compositions were tested for optimized protein loading. RLX2 and O-RLX2 were quantified by HPLC. The capability of the peptides/conjugate to stimulate transfected cells to produce cyclic adenosine monophosphate (cAMP) was used as a measure of their biological activity. The stability and bioactivity of free and liposomal RLX2 types were monitored for a 30 d period, in buffer (in some cases) and bovine serum (80%) at 37 degrees C. The results showed that liposome encapsulation substantially increased the RLX2 integrity in buffer; PEGylated liposomes demonstrated a higher protection. Liposome encapsulation also increased the stability of RLX2 and O-RLX2 in serum. Considering the peptide's biological activity, cAMP production of RLX2 was higher than that of the oxidized form and the P-RLX2 conjugate (which demonstrated a similar activity to O-RLX2 when measured in buffer, but lower when measured in the presence of serum proteins), while liposome encapsulation resulted in a slight decrease of bioactivity initially, but prolonged the peptide bioactivity during incubation in serum. It was concluded that liposome encapsulation of RLX2 and synthetic modification to P-RLX2 can both prolong RLX2 peptide in vitro stability; however, the applied chemical conjugation results in a significant loss of bioactivity (cAMP production), whereas the effect of liposome entrapment on RLX2 activity was significantly lower.
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