Fat grafting, a key regenerative medicine technique, often requires repeat procedures due to high-fat reabsorption and volume loss. Addressing this, a novel drug delivery system uniquely combines a thermosensitive, FDA-approved hydrogel (itaconic acid-modified PLGA-PEG-PLGA copolymer) with FGF2-STAB, a stable fibroblast growth factor 2 with a 21-day stability, far exceeding a few hours of wild-type FGF2's stability. Additionally, the growth factor was encapsulated in "green" liposomes prepared via the Mozafari method, ensuring pH protection. The system, characterized by first-order FGF2-STAB release, employs green chemistry for biocompatibility, bioactivity, and eco-friendliness. The liposomes, with diameters of 85.73 ± 3.85 nm and 68.6 ± 2.2% encapsulation efficiency, allowed controlled FGF2-STAB release from the hydrogel compared to the unencapsulated FGF2-STAB. Yet, the protein compromised the carrier's hydrolytic stability. Prior tests were conducted on model proteins human albumin (efficiency 80.8 ± 3.2%) and lysozyme (efficiency 81.0 ± 2.7%). This injectable thermosensitive system could advance reconstructive medicine and cosmetic procedures.

Central European Institute of Technology Brno University of Technology Purkyňova 656 123 612 00 Brno Czech Republic

Faculty of Chemistry Brno University of Technology Purkyňova 464 612 00 Brno Czech Republic

See more in PubMed

Illouz Y. G.; Sterodimas A. Autologous Fat Transplantation to the Breast: A Personal Technique with 25 Years of Experience. Aesthetic Plast. Surg. 2009, 33 (5), 706–715. 10.1007/s00266-009-9377-1. PubMed DOI

Granoff M. D.; Guo L.; Singhal D. Lipofilling after Breast Conserving Surgery: A Plastic Surgery Perspective. Gland Surg. AME Publishing Company 2020, 9 (3), 617–619. 10.21037/gs.2020.04.02. PubMed DOI PMC

Hörl H. W.; Feller A.-M.; Biemer E. Technique for Liposuction Fat Reimplantation and Long-Term Volume Evaluation by Magnetic Resonance Imaging. Ann. Plast. Surg. 1991, 26 (3), 248–258. 10.1097/00000637-199103000-00007. PubMed DOI

Niechajev I.; Śevćuk O. Long-Term Results of Fat Transplantation. Plast. Reconstr. Surg. 1994, 94 (3), 496–506. 10.1097/00006534-199409000-00012. PubMed DOI

Doornaert M.; Colle J.; De Maere E.; Declercq H.; Blondeel P. Autologous Fat Grafting: Latest Insights. Ann. med. surg. 2019, 37, 47–53. 10.1016/j.amsu.2018.10.016. PubMed DOI PMC

Herly M.; Ørholt M.; Larsen A.; Pipper C. B.; Bredgaard R.; Gramkow C. S.; Katz A. J.; Drzewiecki K. T.; Vester-Glowinski P. V. Efficacy of Breast Reconstruction with Fat Grafting: A Systematic Review and Meta-Analysis. Br. J. Plast. Surg. 2018, 71 (12), 1740–1750. 10.1016/j.bjps.2018.08.024. PubMed DOI

Lui Y. F.; Ip W. Y. Application of Hydrogel in Reconstruction Surgery: Hydrogel/Fat Graft Complex Filler for Volume Reconstruction in Critical Sized Muscle Defects. BioMed Res. Int. 2016, 2016, 3459431.10.1155/2016/3459431. PubMed DOI PMC

Alghoul M.; Mendiola A.; Seth R.; Rubin B. P.; Zins J. E.; Calabro A.; Siemionow M.; Kusuma S. The Effect of Hyaluronan Hydrogel on Fat Graft Survival. Aesthet Surg J. 2012, 32 (5), 622–633. 10.1177/1090820X12448794. PubMed DOI

Kayabolen A.; Keskin D.; Aykan A.; Karslıoglu Y.; Zor F.; Tezcaner A. Native Extracellular Matrix/Fibroin Hydrogels for Adipose Tissue Engineering with Enhanced Vascularization. Biomed Mater. 2017, 12 (3), 035007.10.1088/1748-605X/aa6a63. PubMed DOI

Chamradová I.; Vojtová L.; Michlovská L.; Poláček P.; Jančář J. Rheological Properties of Functionalised Thermosensitive Copolymers for Injectable Applications in Medicine. Chem. Pap. 2012, 66 (10), 977–980. 10.2478/s11696-012-0210-y. DOI

Furtado M.; Chen L.; Chen Z.; Chen A.; Cui W. Development of Fish Collagen in Tissue Regeneration and Drug Delivery. Eng. Regen 2022, 3 (3), 217–231. 10.1016/j.engreg.2022.05.002. DOI

Li K.; Yu L.; Liu X.; Chen C.; Chen Q.; Ding J. A Long-Acting Formulation of a Polypeptide Drug Exenatide in Treatment of Diabetes Using an Injectable Block Copolymer Hydrogel. Biomaterials 2013, 34 (11), 2834–2842. 10.1016/j.biomaterials.2013.01.013. PubMed DOI

Choi S.; Baudys M.; Kim S. W. Control of Blood Glucose by Novel GLP-1 Delivery Using Biodegradable Triblock Copolymer of PLGA-PEG-PLGA in Type 2 Diabetic Rats. Pharm. Res. 2004, 21 (5), 827–831. 10.1023/B:PHAM.0000026435.27086.94. PubMed DOI

Kim B. S.; Cho C. S. Injectable Hydrogels for Regenerative Medicine. Tissue Eng. Regener. Med. 2018, 15 (5), 511–512. 10.1007/s13770-018-0161-7. PubMed DOI PMC

Hines D. J.; Kaplan D. L. Poly(Lactic-Co-Glycolic) Acid-Controlled-Release Systems: Experimental and Modeling Insights. Crit. Rev. Ther. Drug Carrier Syst. 2013, 30 (3), 257–276. 10.1615/CritRevTherDrugCarrierSyst.2013006475. PubMed DOI PMC

Cohen S.; Yoshioka T.; Lucarelli M.; Hwang L. H.; Langer R. Controlled Delivery Systems for Proteins Based on Poly(Lactic/Glycolic Acid) Microspheres. Pharm. Res. 1991, 08 (6), 713–720. 10.1023/A:1015841715384. PubMed DOI

Jain A.; Kunduru K. R.; Basu A.; Mizrahi B.; Domb A. J.; Khan W. Injectable Formulations of Poly(Lactic Acid) and Its Copolymers in Clinical Use. Adv. Drug Delivery Rev. 2016, 107, 213–227. 10.1016/j.addr.2016.07.002. PubMed DOI

Aaron DuVall G.; Tarabar D.; Seidel R. H.; Elstad N. L.; Fowers K. D. Phase 2: A Dose-Escalation Study of OncoGel (ReGel/Paclitaxel), a Controlled-Release Formulation of Paclitaxel, as Adjunctive Local Therapy to External-Beam Radiation in Patients with Inoperable Esophageal Cancer. Anticancer Drugs 2009, 20 (2), 89–95. 10.1097/cad.0b013e3283222c12. PubMed DOI

Elstad N. L.; Fowers K. D. OncoGel (ReGel/Paclitaxel) - Clinical Applications for a Novel Paclitaxel Delivery System. Adv. Drug Delivery Rev. 2009, 61 (10), 785–794. 10.1016/j.addr.2009.04.010. PubMed DOI

Michlovská L.; Vojtová L.; Mravcová L.; Hermanová S.; Kučerík J.; Jančář J. Functionalization Conditions of PLGA-PEG-PLGA Copolymer with Itaconic Anhydride. Macromol. Symp. 2010, 295, 119–124. 10.1002/masy.200900071. DOI

Okabe M.; Lies D.; Kanamasa S.; Park E. Y. Biotechnological Production of Itaconic Acid and Its Biosynthesis in Aspergillus Terreus. Appl. Microbiol. Biotechnol. 2009, 84 (4), 597–606. 10.1007/s00253-009-2132-3. PubMed DOI

Willke T.; Vorlop K. D. Biotechnological Production of Itaconic Acid. Appl. Microbiol. Biotechnol. 2001, 56 (3–4), 289–295. 10.1007/s002530100685. PubMed DOI

Karaffa L.; Kubicek C. P. Citric Acid and Itaconic Acid Accumulation: Variations of the Same Story?. Appl. Microbiol. Biotechnol. 2019, 103 (7), 2889–2902. 10.1007/s00253-018-09607-9. PubMed DOI PMC

Perioli L.; Ambrogi V.; Angelici F.; Ricci M.; Giovagnoli S.; Capuccella M.; Rossi C. Development of Mucoadhesive Patches for Buccal Administration of Ibuprofen. J. Controlled Release 2004, 99 (1), 73–82. 10.1016/j.jconrel.2004.06.005. PubMed DOI

Carvalho F. C.; Bruschi M. L.; Evangelista R. C.; Gremião M. P. D. Mucoadhesive Drug Delivery Systems. Braz. J. Pharm. Sci. 2010, 46 (1), 1–17. 10.1590/S1984-82502010000100002. DOI

Maeda T. Structures and Applications of Thermoresponsive Hydrogels and Nanocomposite-Hydrogels Based on Copolymers with Poly (Ethylene Glycol) and Poly (Lactide-Co-Glycolide) Blocks. Bioeng 2019, 6 (4), 107.10.3390/bioengineering6040107. PubMed DOI PMC

Makadia H. K.; Siegel S. J. Poly Lactic-Co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers 2011, 3 (3), 1377–1397. 10.3390/polym3031377. PubMed DOI PMC

Oborna J.; Mravcova L.; Michlovska L.; Vojtova L.; Vavrova M. The Effect of PLGA-PEG-PLGA Modification on the Sol-Gel Transition and Degradation Properties. eXPRESS Polym. Lett. 2016, 10 (5), 361–372. 10.3144/expresspolymlett.2016.34. DOI

Qiao M.; Chen D.; Hao T.; Zhao X.; Hu H.; Ma X. Injectable Thermosensitive PLGA-PEG-PLGA Triblock Copolymers-Based Hydrogels as Carriers for Interleukin-2. Pharmazie 2008, 63 (1), 27–30. PubMed

Chen Y.; Shi J.; Zhang Y.; Miao J.; Zhao Z.; Jin X.; Liu L.; Yu L.; Shen C.; Ding J. An Injectable Thermosensitive Hydrogel Loaded with an Ancient Natural Drug Colchicine for Myocardial Repair after Infarction. J. Mater. Chem. B 2020, 8 (5), 980–992. 10.1039/C9TB02523E. PubMed DOI

Akasaka Y.; Ono I.; Kamiya T.; Ishikawa Y.; Kinoshita T.; Ishiguro S.; Yokoo T.; Imaizumi R.; Inomata N.; Fujita K.; Akishima-Fukasawa Y.; Uzuki M.; Ito K.; Ishii T. The Mechanisms Underlying Fibroblast Apoptosis Regulated by Growth Factors during Wound Healing. J. Pathol. 2010, 221 (3), 285–299. 10.1002/path.2710. PubMed DOI

Nugent M. A.; Iozzo R. V. Fibroblast Growth Factor-2. Int. J. Biochem. Cell Biol. 2000, 32, 115–120. 10.1016/S1357-2725(99)00123-5. PubMed DOI

Dvorak P.; Bednar D.; Vanacek P.; Balek L.; Eiselleova L.; Stepankova V.; Sebestova E.; Kunova Bosakova M.; Konecna Z.; Mazurenko S.; Kunka A.; Vanova T.; Zoufalova K.; Chaloupkova R.; Brezovsky J.; Krejci P.; Prokop Z.; Dvorak P.; Damborsky J. Computer-Assisted Engineering of Hyperstable Fibroblast Growth Factor 2. Biotechnol. Bioeng. 2018, 115 (4), 850–862. 10.1002/bit.26531. PubMed DOI

Buchtova M.; Chaloupkova R.; Zakrzewska M.; Vesela I.; Cela P.; Barathova J.; Gudernova I.; Zajickova R.; Trantirek L.; Martin J.; Kostas M.; Otlewski J.; Damborsky J.; Kozubik A.; Wiedlocha A.; Krejci P. Instability Restricts Signaling of Multiple Fibroblast Growth Factors. Cell. Mol. Life Sci. 2015, 72, 2445–2459. 10.1007/s00018-015-1856-8. PubMed DOI PMC

Lysakova K.; Hlinakova K.; Kutalkova K.; Chaloupkova R.; Zidek J.; Brtnikova J.; Vojtova L. Novel approach in control release monitoring of protein-based bioactive substances from injectable PLGA-PEG-PLGA hydrogel. eXPRESS Polym. Lett. 2022, 16 (8), 798–811. 10.3144/expresspolymlett.2022.59. DOI

Tanford C. Protein Denaturation. Adv. Protein Chem. Struct. Biol. 1968, 23, 121–282. 10.1016/S0065-3233(08)60401-5. PubMed DOI

M., Reza Mozafari. Method for the Preparation of Micro- and Nano-Sized Carrier Systems for the Encapsulation of Bioactive Substances, 2010, U.S. Patent 20,100,239,521 A1.

Maleki G.; Bahrami Z.; Woltering E. J.; Khorasani S.; Mozafari M. R. A Review of Patents on “Mozafari Method” as a Green Technology for Manufacturing Bioactive Carriers. Biointerface Res. Appl. Chem. 2023, 13 (1), 34–49.

Mortazavi S. M.; Mohammadabadi M. R.; Khosravi-Darani K.; Mozafari M. R. Preparation of Liposomal Gene Therapy Vectors by a Scalable Method without Using Volatile Solvents or Detergents. J. Biotechnol. 2007, 129 (4), 604–613. 10.1016/j.jbiotec.2007.02.005. PubMed DOI

Mosharraf M.; Taylor K. M. G.; Craig D. Q. M. Effect of Calcium Ions on the Surface Charge and Aggregation of Phosphatidylcholine Liposomes. J. Drug Targeting 1995, 2 (6), 541–545. 10.3109/10611869509015925. PubMed DOI

Cavalcanti I. M. F.; Mendonça E. A.; Lira M. C. B.; Honrato S. B.; Camara C. A.; Amorim R. V. S.; Filho J. M.; Rabello M. M.; Hernandes M. Z.; Ayala A. P.; Santos-Magalhães N. S. The Encapsulation of β-Lapachone in 2-Hydroxypropyl-β-Cyclodextrin Inclusion Complex into Liposomes: A Physicochemical Evaluation and Molecular Modeling Approach. Eur. J. Pharm. Sci. 2011, 44 (3), 332–340. 10.1016/j.ejps.2011.08.011. PubMed DOI

Xu H. L.; Chen P. P.; Wang L. f.; Xue W.; Fu T. L. Hair Regenerative Effect of Silk Fibroin Hydrogel with Incorporation of FGF-2-Liposome and Its Potential Mechanism in Mice with Testosterone-Induced Alopecia Areata. J. Drug Delivery Sci. Technol. 2018, 48, 128–136. 10.1016/j.jddst.2018.09.006. DOI

Jahadi M.; Khosravi-Darani K.; Ehsani M. R.; Mozafari M. R.; Saboury A. A.; Pourhosseini P. S. The Encapsulation of Flavourzyme in Nanoliposome by Heating Method. J. Food Sci. Technol. 2015, 52 (4), 2063–2072. 10.1007/s13197-013-1243-0. PubMed DOI PMC

Jahanfar S.; Gahavami M.; Khosravi-Darani K.; Jahadi M.; Mozafari M. R. Entrapment of Rosemary Extract by Liposomes Formulated by Mozafari Method: Physicochemical Characterization and Optimization. Heliyon 2021, 7 (12), e0863210.1016/j.heliyon.2021.e08632. PubMed DOI PMC

Mozafari M. R. R.; Reed C. J.; Rostron C. Development of Non-Toxic Liposomal Formulations for Gene and Drug Delivery to the Lung. Technol. Health Care 2002, 10 (3–4), 342–344.

Awad R. S.; Abdelwahed W.; Bitar Y. Evaluating the Impact of Preparation Conditions and Formulation on the Accelerated Stability of Tretinoin Loaded Liposomes Prepared by Heating Method. Int. J. Pharm. Sci. 2015, 7 (5), 171–178.

Manca M. L.; Peris J. E.; Melis V.; Valenti D.; Cardia M. C.; Lattuada D.; Escribano-Ferrer E.; Fadda A. M.; Manconi M. Nanoincorporation of Curcumin in Polymer-Glycerosomes and Evaluation of Their in Vitro-in Vivo Suitability as Pulmonary Delivery Systems. RSC Adv. 2015, 5 (127), 105149–105159. 10.1039/C5RA24032H. DOI

Zhang K.; Zhang Y.; Li Z.; Li N.; Feng N. Essential Oil-Mediated Glycerosomes Increase Transdermal Paeoniflorin Delivery: Optimization, Characterization, and Evaluation in Vitro and in Vivo. Int. J. Nanomed. 2017, 12, 3521–3532. 10.2147/IJN.S135749. PubMed DOI PMC

Poudel A.; Gachumi G.; Wasan K. M.; Dallal Bashi Z.; El Aneed A.; Badea I. Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives. Pharmaceutics 2019, 11 (4), 185.10.3390/pharmaceutics11040185. PubMed DOI PMC

Wu H.; Cao D.; Liu T.; Zhao J.; Hu X.; Li N. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens. PLoS One 2015, 10 (12), e014603210.1371/journal.pone.0146032. PubMed DOI PMC

Refetoff S.Thyroid Hormone Transport Proteins: Thyroxine-Binding Globulin, Transthyretin, and Albumin. In Encyclopedia of Hormones, 1st ed., Henry H. L., Norman A. W., Eds.; Elsevier Academic Press: San Diego, CA, 2003; pp 483–490.

Castellanos M. M.; Colina C. M. Molecular Dynamics Simulations of Human Serum Albumin and Role of Disulfide Bonds. J. Phys. Chem. B 2013, 117 (40), 11895–11905. 10.1021/jp402994r. PubMed DOI

Bernsdorff C.; Reszka R.; Winter R. Interaction of the Anticancer Agent TaxolTM (Paclitaxel) with Phospholipid Bilayers. J. Biomed. Mater. Res. 1999, 46 (2), 141–149. 10.1002/(SICI)1097-4636(199908)46:2<141::AID-JBM2>3.0.CO;2-U. PubMed DOI

Al-Ayed M. S. Biophysical Studies on the Liposome-Albumin System. Indian J. Biochem. Biphys. 2006, 43 (3), 186–189. PubMed

Jafari M.; Mehrnejad F. Molecular Insight into Human Lysozyme and Its Ability to Form Amyloid Fibrils in High Concentrations of Sodium Dodecyl Sulfate: A View from Molecular Dynamics Simulations. PLoS One 2016, 11 (10), e016521310.1371/journal.pone.0165213. PubMed DOI PMC

Lopes N. A.; Barreto Pinilla C. M.; Brandelli A. Antimicrobial Activity of Lysozyme-Nisin Co-Encapsulated in Liposomes Coated with Polysaccharides. Food Hydrocolloids 2019, 93, 1–9. 10.1016/j.foodhyd.2019.02.009. DOI

Xu H.-L.; Chen P.-P.; ZhuGe D.-L.; Zhu Q.-Y.; Jin B.-H.; Shen B.-X.; Xiao J.; Zhao Y.-Z. Liposomes with Silk Fibroin Hydrogel Core to Stabilize BFGF and Promote the Wound Healing of Mice with Deep Second-Degree Scald. Adv. Healthcare Mater. 2017, 6 (19), 1700344.10.1002/adhm.201700344. PubMed DOI

Michlovská L.; Vojtová L.; Humpa O.; Kučerík J.; Žídek J.; Jančář J. Hydrolytic Stability of End-Linked Hydrogels from PLGA-PEG-PLGA Macromonomers Terminated by α,ω-Itaconyl Groups. RSC Adv. 2016, 6 (20), 16808–16816. 10.1039/C5RA26222D. DOI

Larkin P. J.Illustrated IR and Raman Spectra Demonstrating Important Functional Groups. In Infrared and Raman Spectroscopy, 2nd ed.; Larkin P. J., Ed.; Elsevier Academic Press: San Diego, CA, 2018; pp 153–210.

Macdonald M. L.; Rodriguez N. M.; Shah N. J.; Hammond P. T. Characterization of Tunable FGF-2 Releasing Polyelectrolyte Multilayers. Biomacromolecules 2010, 11 (8), 2053–2059. 10.1021/bm100413w. PubMed DOI PMC

Wiig H.; Kolmannskog O.; Tenstad O.; Bert J. L. Effect of Charge on Interstitial Distribution of Albumin in Rat Dermis in Vitro. J. Physiol. 2003, 550 (2), 505–514. 10.1113/jphysiol.2003.042713. PubMed DOI PMC

Paarakh M.; Jose P.; Setty C.; Christoper G. RELEASE KINETICS - CONCEPTS AND APPLICATIONS. Int. J. Pharm. Technol. 2019, 8 (1), 220279629.10.31838/ijprt/08.01.02. DOI

Cao D.; Zhang X.; Akabar M. D.; Luo Y.; Wu H.; Ke X.; Ci T. Liposomal Doxorubicin Loaded PLGA-PEG-PLGA Based Thermogel for Sustained Local Drug Delivery for the Treatment of Breast Cancer. Artif. Cells, Nanomed., Biotechnol. 2019, 47 (1), 181–191. 10.1080/21691401.2018.1548470. PubMed DOI