{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T19:27:27Z","timestamp":1773343647699,"version":"3.50.1"},"reference-count":36,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2022,9,24]],"date-time":"2022-09-24T00:00:00Z","timestamp":1663977600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Management Authority of Western Greece Region and the Public Investment Programme of Greece","award":["81762"],"award-info":[{"award-number":["81762"]}]},{"name":"MEDICUS","award":["81762"],"award-info":[{"award-number":["81762"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Relaxin (RLX) is a protein that is structurally similar to insulin and has interesting biological activities. As with all proteins, preservation of RLX\u2019s structural integrity\/biological functionality is problematic. Herein, we investigated two methods for increasing the duration of relaxin-2\u2019s (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 \u00b0C. 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\u2019s 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.<\/jats:p>","DOI":"10.3390\/biom12101362","type":"journal-article","created":{"date-parts":[[2022,9,25]],"date-time":"2022-09-25T21:14:28Z","timestamp":1664140468000},"page":"1362","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Liposomal Entrapment or Chemical Modification of Relaxin2 for Prolongation of Its Stability and Biological Activity"],"prefix":"10.3390","volume":"12","author":[{"given":"George","family":"Kogkos","sequence":"first","affiliation":[{"name":"Lab Pharm Technology, Department of Pharmacy, University of Patras, Rio, 26504 Patras, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8605-9895","authenticated-orcid":false,"given":"Foteini","family":"Gkartziou","sequence":"additional","affiliation":[{"name":"Foundation for Research and Technology Hellas, Institute of Chemical Engineering, FORTH\/ICE-HT, Platani, 26504 Patras, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1204-1574","authenticated-orcid":false,"given":"Spyridon","family":"Mourtas","sequence":"additional","affiliation":[{"name":"Lab Pharm Technology, Department of Pharmacy, University of Patras, Rio, 26504 Patras, Greece"},{"name":"Department of Chemistry, University of Patras, Rio, 26504 Patras, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kostas K.","family":"Barlos","sequence":"additional","affiliation":[{"name":"Chemical & Biopharmaceutical Laboratories CBL Patras, Ind. Area of Patras, Block 1, 25018 Patras, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pavlos","family":"Klepetsanis","sequence":"additional","affiliation":[{"name":"Lab Pharm Technology, Department of Pharmacy, University of Patras, Rio, 26504 Patras, Greece"},{"name":"Foundation for Research and Technology Hellas, Institute of Chemical Engineering, FORTH\/ICE-HT, Platani, 26504 Patras, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kleomenis","family":"Barlos","sequence":"additional","affiliation":[{"name":"Chemical & Biopharmaceutical Laboratories CBL Patras, Ind. Area of Patras, Block 1, 25018 Patras, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2312-5848","authenticated-orcid":false,"given":"Sophia G.","family":"Antimisiaris","sequence":"additional","affiliation":[{"name":"Lab Pharm Technology, Department of Pharmacy, University of Patras, Rio, 26504 Patras, Greece"},{"name":"Foundation for Research and Technology Hellas, Institute of Chemical Engineering, FORTH\/ICE-HT, Platani, 26504 Patras, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"950","DOI":"10.1111\/bph.13684","article-title":"Relaxin family peptides: Structure-activity relationship studies","volume":"174","author":"Patil","year":"2017","journal-title":"Br. J. Pharmacol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1124\/pr.58.1.9","article-title":"International union of pharmacology LVII: Recommendations for the nomenclature of receptors for relaxin family peptides","volume":"58","author":"Bathgate","year":"2006","journal-title":"Pharmacol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2739","DOI":"10.1172\/JCI119099","article-title":"Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo","volume":"98","author":"Unemori","year":"1996","journal-title":"J. Clin. Investig."},{"key":"ref_4","first-page":"227","article-title":"A preliminary investigation of the effect of relaxin on bone remodelling in suture expansion","volume":"39","author":"Duarte","year":"2017","journal-title":"Eur. J. Orthod."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/S0140-6736(12)61855-8","article-title":"RELAXin in Acute Heart Failure (RELAX-AHF) Investigators. Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): A randomised, placebo-controlled trial","volume":"381","author":"Teerlink","year":"2013","journal-title":"Lancet"},{"key":"ref_6","first-page":"247","article-title":"Stability and characterization of recombinant human relaxin","volume":"1996 9","author":"Nguyen","year":"1996","journal-title":"Pharm. Biotechnol."},{"key":"ref_7","unstructured":"Pearlman, R., and Wangs, Y.J. (1996). Stability and Characterization of Recombinant Human Relaxin. Formulation, Characterization, and Stability of Protein Drugs, Kluwer Academic\/Plenum Publishers."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1002\/psc.1221","article-title":"An optimized chemical synthesis of human relaxin-2","volume":"16","author":"Barlos","year":"2010","journal-title":"J. Pept. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1325","DOI":"10.1023\/A:1025771421906","article-title":"Physical stability of proteins in aqueous solution: Mechanism and driving forces in nonnative protein aggregation","volume":"20","author":"Chi","year":"2003","journal-title":"Pharm. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1023\/A:1018982726441","article-title":"The pharmacokinetics and absorption of recombinant human relaxin in nonpregnant rabbits and rhesus monkeys after intravenous and intravaginal administration","volume":"10","author":"Chen","year":"1993","journal-title":"Pharm. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1023\/A:1018901009062","article-title":"The pharmacokinetics of recombinant human relaxin in nonpregnant women after intravenous, intravaginal, and intracervical administration","volume":"10","author":"Chen","year":"1993","journal-title":"Pharm. Res."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Wijesinghe, A., Kumari, S., and Booth, V. (2022). Conjugates for use in peptide therapeutics: A systematic review and meta-analysis. PLoS ONE, 17.","DOI":"10.1371\/journal.pone.0255753"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"10895","DOI":"10.1039\/C5OB01539A","article-title":"Chemically synthesized dicarba H2 relaxin analogues retain strong RXFP1 receptor activity but show an unexpected loss of in vitro serum stability","volume":"13","author":"Hossain","year":"2015","journal-title":"Org. Biomol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Vinther, T.N., Norrman, M., Strauss, H.M., Huus, K., Schlein, M., Pedersen, T.\u00c5., Kjeldsen, T., Jensen, K.J., and Hub\u00e1lek, F. (2012). Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0030882"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"731852","DOI":"10.1155\/2015\/731852","article-title":"Synthetic covalently linked dimeric form of H2 relaxin retains native RXFP1 activity and has improved in vitro serum stability","volume":"2015","author":"Nair","year":"2015","journal-title":"Biomed. Res. Int."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1002\/9780470110546.ch7","article-title":"Liposomes: Preparation, characterization and preservation","volume":"33","author":"Lichtenberg","year":"1998","journal-title":"Methods Biochem. Anal."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"11443","DOI":"10.1073\/pnas.93.21.11443","article-title":"Rapid preparation of giant unilamellar vesicles","volume":"93","author":"Moscho","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1111\/j.1470-8744.1999.tb00774.x","article-title":"Butyrylcholinesterase formulated in liposomes","volume":"30","author":"Schumacher","year":"1999","journal-title":"Biotechnol. Appl. Biochem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1081\/BIO-120028669","article-title":"Encapsulation of enzymes in liposomes: High encapsulation effi-ciency and control of substrate permeability","volume":"32","author":"Chaize","year":"2004","journal-title":"Artif. Cells Blood Substit. Immobil. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1002\/bit.10074","article-title":"Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation","volume":"75","author":"Nasseau","year":"2001","journal-title":"Biotechnol. Bioeng."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1007\/978-3-319-66095-0_9","article-title":"Brimble Peptide Lipidation\u2014A Synthetic Strategy to Afford Peptide Based Therapeutics","volume":"1030","author":"Kowalczyk","year":"2017","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1038\/nbt1184-979","article-title":"Dehydration-rehydration vesicles: A simple method for high yield drug entrapment in liposomes","volume":"2","author":"Kirby","year":"1984","journal-title":"Nat. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/0378-5173(90)90148-W","article-title":"Florence. A procedure for the efficient entrapment of drugs in dehydration-rehydration liposomes (DRVs)","volume":"65","author":"Gregoriadis","year":"1990","journal-title":"Int. J. Pharm."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"14788","DOI":"10.1038\/s41598-021-94130-7","article-title":"Synergistic effect of cold atmospheric pressure plasma and free or liposomal doxorubicin on melanoma cells","volume":"11","author":"Athanasopoulos","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"542","DOI":"10.3390\/pharmaceutics5040542","article-title":"Surface engineering of liposomes for stealth behavior","volume":"5","author":"Nag","year":"2013","journal-title":"Pharmaceutics"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/0003-2697(80)90269-9","article-title":"Colorimetric determination of phospholipids with ammonium ferrothiocyanate","volume":"104","author":"Stewart","year":"1980","journal-title":"Anal. Biochem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/j.ijpharm.2018.11.060","article-title":"Rapid and scale-independent microfluidic manufacture of liposomes entrapping protein incorporating in-line purification and at-line size monitoring","volume":"556","author":"Forbes","year":"2019","journal-title":"Int. J. Pharm."},{"key":"ref_28","first-page":"15","article-title":"Optimization of a Method to Prepare Liposomes Containing HER2\/Neu-Derived Peptide as a Vaccine Delivery System for Breast Cancer","volume":"13","author":"Shariat","year":"2014","journal-title":"Iran. J. Pharm. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2419","DOI":"10.1210\/en.2008-1385","article-title":"Characterization of relaxin receptor (RXFP1) desensitization and internalization in primary human decidual cells and RXFP1-transfected HEK293 cells","volume":"150","author":"Kern","year":"2009","journal-title":"Endocrinology"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.chemphyslip.2005.11.003","article-title":"Incorporation of PEG-lipids in arsonoliposomes results in formation of highly stable arsenic-containing vesicles","volume":"139","author":"Piperoudi","year":"2006","journal-title":"Chem. Phys. Lipids"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Natsaridis, E., Gkartziou, F., Mourtas, S., Stuart, M.C.A., Kolonitsiou, F., Klepetsanis, P., Spiliopoulou, I., and Antimisiaris, S.G. (2022). Moxifloxacin Liposomes: Effect of Liposome Preparation Method on Physicochemical Properties and Antimicrobial Activity against Staphylococcus epidermidis. Pharmaceutics, 14.","DOI":"10.3390\/pharmaceutics14020370"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.ijpharm.2014.09.041","article-title":"Increasing the stability of curcumin in serum with liposomes or hybrid drug-in-cyclodextrin-in-liposome systems: A comparative study","volume":"476","author":"Matloob","year":"2014","journal-title":"Int. J. Pharm."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5773","DOI":"10.1021\/la804215v","article-title":"Effective stabilization of curcumin by association to plasma proteins: Human serum albumin and fibrinogen","volume":"25","author":"Leung","year":"2009","journal-title":"Langmuir"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/1472-6750-2-9","article-title":"Protein encapsulation in liposomes: Efficiency depends on interactions between protein and phospholipid bilayer","volume":"2","author":"Colletier","year":"2002","journal-title":"BMC Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.ijpharm.2012.04.057","article-title":"Haemocompatibility improvement of metallic surfaces by covalent immobilization of heparin-liposomes","volume":"432","author":"Kastellorizios","year":"2012","journal-title":"Int. J. Pharm."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"891","DOI":"10.1021\/cb500918v","article-title":"Solution Structure, Aggregation Behavior, and Flexibility of Human Relaxin-2","volume":"10","author":"Hossain","year":"2015","journal-title":"ACS Chem. Biol."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/12\/10\/1362\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:38:36Z","timestamp":1760143116000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/12\/10\/1362"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,24]]},"references-count":36,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2022,10]]}},"alternative-id":["biom12101362"],"URL":"https:\/\/doi.org\/10.3390\/biom12101362","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,24]]}}}