Liposomal Entrapment or Chemical Modification of Relaxin2 for Prolongation of Its Stability and Biological Activity

George Kogkos; Foteini Gkartziou; Spyridon Mourtas; Kostas K Barlos; Pavlos Klepetsanis; Kleomenis Barlos; Sophia G Antimisiaris

doi:10.3390/biom12101362

Liposomal Entrapment or Chemical Modification of Relaxin2 for Prolongation of Its Stability and Biological Activity

Biomolecules. 2022 Sep 24;12(10):1362. doi: 10.3390/biom12101362.

Authors

George Kogkos¹, Foteini Gkartziou², Spyridon Mourtas^{1

3}, Kostas K Barlos⁴, Pavlos Klepetsanis^{1

2}, Kleomenis Barlos⁴, Sophia G Antimisiaris^{1

2}

Affiliations

¹ Lab Pharm Technology, Department of Pharmacy, University of Patras, Rio, 26504 Patras, Greece.
² Foundation for Research and Technology Hellas, Institute of Chemical Engineering, FORTH/ICE-HT, Platani, 26504 Patras, Greece.
³ Department of Chemistry, University of Patras, Rio, 26504 Patras, Greece.
⁴ Chemical & Biopharmaceutical Laboratories CBL Patras, Ind. Area of Patras, Block 1, 25018 Patras, Greece.

Abstract

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 °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.

Keywords: biological activity; lipidic conjugate; liposomes; peptides; relaxin; stability.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Monophosphate
Insulins*
Lipids
Liposomes / chemistry
Polyethylene Glycols
Relaxin*

Substances

Liposomes
Relaxin
Polyethylene Glycols
Lipids
Adenosine Monophosphate
Insulins

Grants and funding

This project was implemented within the context of the research program entitled “Development of Innovative Nanocarriers of Insulin-like Peptides with prolonged action, NANO INS” MIS 5021443, which is co-funded by the Management Authority of Western Greece Region and the Public Investment Programme of Greece. SM received funding from the programme “MEDICUS” of the University of Patras –Research Committee, Code 81762.