{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T14:17:45Z","timestamp":1781878665749,"version":"3.54.5"},"reference-count":45,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2021,12,5]],"date-time":"2021-12-05T00:00:00Z","timestamp":1638662400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Fondi di Ateneo 2021 to M.C.B. and D.E.P-G","award":["MARIACAMILLABERGONZIRICATEN21"],"award-info":[{"award-number":["MARIACAMILLABERGONZIRICATEN21"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Thymoquinone (TQ) is the main constituent of Nigella sativa L. essential oil. In vitro studies have shown its protective effect against H2O2-induced oxidative stress in human retinal pigment epithelium cells, and in vivo experiments have demonstrated its effect in decreasing corneal neovascularization and reducing the inflammation in an experimental dry eye model in mice. Its therapeutic use is limited by poor bioavailability, low solubility, and scarce permeability. In this study, two liposomal formulations have been developed, both of which consist of phosphatidylcholine and Plurol Oleique, a liquid lipid, and one of which is coated with 0.1% w\/v hyaluronic acid (HA) to increase both TQ solubility and its ocular therapeutic potential. Each formulation has a size &lt;200 nm and an EE% around 70%, determined by scattering techniques and the HPLC-DAD analytical method, respectively, and they result in a 2-fold increase in TQ solubility. HA-coated liposomes are stable over 2 months at +4 \u00b0C, and coated and uncoated liposomes present a gradual and prolonged release of TQ. Two cell lines, human corneal epithelial cells (HCEC-2) and human conjunctival epithelial cells (HConEC) were used to investigate the safety of the liposomal formulations. Uptake studies were also performed using fluorescent liposomes. Both liposomes and, in particular, HA-coated liposomes reduce the TQ toxicity observed at high doses in both HCEC-2 and HConEC cells, and both formulations increase the absorption at the cellular level and especially at the nucleus level, with a more pronounced effect for HA-coated liposomes.<\/jats:p>","DOI":"10.3390\/pharmaceutics13122093","type":"journal-article","created":{"date-parts":[[2021,12,6]],"date-time":"2021-12-06T03:10:38Z","timestamp":1638760238000},"page":"2093","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":27,"title":["Preparation of Liposomal Formulations for Ocular Delivery of Thymoquinone: In Vitro Evaluation in HCEC-2 e HConEC Cells"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6776-2857","authenticated-orcid":false,"given":"Elisa","family":"Landucci","sequence":"first","affiliation":[{"name":"Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Bonomolo","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florence, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8165-6607","authenticated-orcid":false,"given":"Chiara","family":"De Stefani","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florence, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Costanza","family":"Mazzantini","sequence":"additional","affiliation":[{"name":"Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Domenico Edoardo","family":"Pellegrini-Giampietro","sequence":"additional","affiliation":[{"name":"Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8772-1895","authenticated-orcid":false,"given":"Anna Rita","family":"Bilia","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florence, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5144-1330","authenticated-orcid":false,"given":"Maria Camilla","family":"Bergonzi","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Florence, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,12,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1528635","DOI":"10.1155\/2019\/1528635","article-title":"Nigella sativa L. (Black Cumin): A Promising Natural Remedy for Wide Range of Illnesses","volume":"2019","author":"Yimer","year":"2019","journal-title":"Evid.-Based Complement. Altern. Med."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Gupta, R.C. (2016). Chapter 39\u2014Thymoquinone. Nutraceuticals, Academic Press. [1st ed.].","DOI":"10.1016\/B978-0-12-802147-7.00039-5"},{"key":"ref_3","unstructured":"Gupta, R.C., Lall, R., and Srivastava, A. (2021). Chapter 52\u2014Thymoquinone. Nutraceuticals, Academic Press. [2nd ed.]."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Farkhondeh, T., Samarghandian, S., Shahri, A.M.P., and Samini, F. (2018). The Neuroprotective Effects of Thymoquinone: A Review. Dose-Response, 16.","DOI":"10.1177\/1559325818761455"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"El-Far, A.H., Al Jaouni, S.K., Li, W., and Mousa, S.A. (2018). Protective Roles of Thymoquinone Nanoformulations: Potential Nanonutraceuticals in Human Diseases. Nutrients, 10.","DOI":"10.3390\/nu10101369"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4514","DOI":"10.1002\/jcb.27739","article-title":"Thymoquinone protects human retinal pigment epithelial cells against hydrogen peroxide induced oxidative stress and apoptosis","volume":"120","author":"Hu","year":"2019","journal-title":"J. Cell. Biochem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"281","DOI":"10.4274\/tjo.50146","article-title":"Effects of Topical Thymoquinone in an Experimental Dry Eye Model","volume":"48","author":"Erkan","year":"2018","journal-title":"Turk. J. Ophthalmol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1749","DOI":"10.1016\/j.intimp.2005.06.008","article-title":"Immunomodulatory and therapeutic properties of the Nigella sativa L. seed","volume":"5","author":"Salem","year":"2005","journal-title":"Int. Immunopharmacol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"591","DOI":"10.3109\/02713683.2011.573898","article-title":"Ameliorative Effect of Thymoquinone on Ovalbumin-induced Allergic Conjunctivitis in Balb\/c Mice","volume":"36","author":"Hayat","year":"2011","journal-title":"Curr. Eye Res."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Landucci, E., Mazzantini, C., Buonvicino, D., Pellegrini-Giampietro, D., and Bergonzi, M. (2021). Neuroprotective Effects of Thymoquinone by the Modulation of ER Stress and Apoptotic Pathway in In Vitro Model of Excitotoxicity. Molecules, 26.","DOI":"10.3390\/molecules26061592"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Bergonzi, M.C., Vasarri, M., Marroncini, G., Barletta, E., and Degl\u2019Innocenti, D. (2020). Thymoquinone-Loaded Soluplus\u00ae-Solutol\u00ae HS15 Mixed Micelles: Preparation, In Vitro Characterization, and Effect on the SH-SY5Y Cell Migration. Molecules, 25.","DOI":"10.3390\/molecules25204707"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"4631","DOI":"10.2174\/0929867325666181101110050","article-title":"Nanocarriers: A Successful Tool to Increase Solubility, Stability and Optimise Bioefficacy of Natural Constituents","volume":"26","author":"Bilia","year":"2019","journal-title":"Curr. Med. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1075","DOI":"10.3109\/10717544.2014.943336","article-title":"Liposomes in topical ophthalmic drug delivery: An update","volume":"23","author":"Agarwal","year":"2014","journal-title":"Drug Deliv."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"765","DOI":"10.3109\/03639045.2013.783589","article-title":"Liposomal diltiazem HCl as ocular drug delivery system for glaucoma","volume":"40","author":"Ibrahim","year":"2014","journal-title":"Drug Dev. Ind. Pharm."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1599","DOI":"10.3109\/03639045.2012.736515","article-title":"Recent advances in ocular drug delivery","volume":"39","author":"Achouri","year":"2013","journal-title":"Drug Dev. Ind. Pharm."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.jconrel.2017.07.035","article-title":"Novel nanosystems for the treatment of ocular inflammation: Current paradigms and future research directions","volume":"268","author":"Lalu","year":"2017","journal-title":"J. Control. Release"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1016\/j.ijpharm.2018.07.012","article-title":"Treatment merits of Latanoprost\/Thymoquinone\u2014Encapsulated liposome for glaucomatus rabbits","volume":"548","author":"Fahmy","year":"2018","journal-title":"Int. J. Pharm."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.addr.2021.01.019","article-title":"Overcoming barriers by local drug delivery with liposomes","volume":"174","author":"Antimisiaris","year":"2021","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_19","first-page":"165","article-title":"Hyaluronan receptors in the human ocular surface: A descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples","volume":"137","author":"Maldonado","year":"2011","journal-title":"Histochem. Cell Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1002\/sctm.18-0178","article-title":"Corneal Wound Healing Effects of Mesenchymal Stem Cell Secretome Delivered Within a Viscoelastic Gel Carrier","volume":"8","author":"Na","year":"2019","journal-title":"Stem Cells Transl. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"142","DOI":"10.2174\/1567201815666181008153602","article-title":"Solid Lipid Nanoparticles and Chitosan-coated Solid Lipid Nanoparticles as Promising Tool for Silybin Delivery: Formulation, Characterization, and In vitro Evaluation","volume":"16","author":"Piazzini","year":"2018","journal-title":"Curr. Drug Deliv."},{"key":"ref_22","first-page":"549","article-title":"Liposomes\u2014Methodology and applications","volume":"48","author":"Ryman","year":"1979","journal-title":"Front. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.colsurfb.2013.12.019","article-title":"Strategy to provide a useful solution to effective delivery of dihydroartemisinin: Development, characterization and in vitro studies of liposomal formulations","volume":"116","author":"Righeschi","year":"2014","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1127","DOI":"10.1007\/s00249-009-0524-z","article-title":"Biophysical studies on chitosan-coated liposomes","volume":"38","author":"Mady","year":"2009","journal-title":"Eur. Biophys. J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"108527","DOI":"10.1016\/j.exer.2021.108527","article-title":"Co-expression of the SARS-CoV-2 entry receptors ACE2 and TMPRSS2 in healthy human conjunctiva","volume":"205","author":"Mencucci","year":"2021","journal-title":"Exp. Eye Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"108269","DOI":"10.1016\/j.exer.2020.108269","article-title":"A new ophthalmic formulation containing antiseptics and dexpanthenol: In vitro antimicrobial activity and effects on corneal and conjunctival epithelial cells","volume":"201","author":"Mencucci","year":"2020","journal-title":"Exp. Eye Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/j.ijpharm.2015.02.013","article-title":"Effects of triglycerides on the hydrophobic drug loading capacity of saturated phosphatidylcholine-based liposomes","volume":"483","author":"Hong","year":"2015","journal-title":"Int. J. Pharm."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2808","DOI":"10.1111\/j.1365-2621.2002.tb08820.x","article-title":"Properties of Liposomes Prepared with Various Lipids","volume":"67","author":"Hsieh","year":"2002","journal-title":"J. Food Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.ijpharm.2016.03.018","article-title":"Percutaneous absorption of benzophenone-3 loaded lipid nanoparticles and polymeric nanocapsules: A comparative study","volume":"504","author":"Gilbert","year":"2016","journal-title":"Int. J. Pharm."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Nasr, A.M., Qushawy, M.K., Elkhoudary, M.M., Gawish, A.Y., Elhady, S.S., and Swidan, S.A. (2020). Quality by Design for the Development and Analysis of Enhanced In-Situ Forming Vesicles for the Improvement of the Bioavailability of Fexofenadine HCl In Vitro and In Vivo. Pharmaceutics, 12.","DOI":"10.3390\/pharmaceutics12050409"},{"key":"ref_31","first-page":"195","article-title":"Microemulsion Drug Delivery System: For Oral Bioavailability Enhancement of Glipizide","volume":"1","author":"Sarkar","year":"2011","journal-title":"J. Adv. Pharm. Educ. Res."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Yeo, L.K., Chaw, C.S., and Elkordy, A.A. (2019). The Effects of Hydration Parameters and Co-Surfactants on Methylene Blue-Loaded Niosomes Prepared by the Thin Film Hydration Method. Pharmaceutics, 12.","DOI":"10.3390\/ph12020046"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.colsurfb.2016.01.014","article-title":"Hyaluronic acid-coated niosomes facilitate tacrolimus ocular delivery: Mucoadhesion, precorneal retention, aqueous humor pharmacokinetics, and transcorneal permeability","volume":"141","author":"Zeng","year":"2016","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1016\/0141-8130(94)90028-0","article-title":"Interactions of hyaluronan (hyaluronic acid) with phospholipids as determined by gel permeation chromatography, multi-angle laser-light-scattering photometry and 1H-NMR spectroscopy","volume":"16","author":"Ghosh","year":"1994","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.ejps.2008.02.003","article-title":"Morphology, structure and supramolecular organization of hybrid 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine\u2013hyaluronic acid microparticles prepared by spray drying","volume":"34","author":"Gaete","year":"2008","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1016\/j.bbamem.2004.10.007","article-title":"Interactions of lipid monolayers with the natural biopolymer hyaluronic acid","volume":"1667","author":"Ionov","year":"2004","journal-title":"Biochim. Biophys. Acta (BBA)-Biomembr."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/j.ejpb.2008.04.025","article-title":"A novel poloxamers\/hyaluronic acid in situ forming hydrogel for drug delivery: Rheological, mucoadhesive and in vitro release properties","volume":"70","author":"Mayol","year":"2008","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/j.jconrel.2016.02.013","article-title":"Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear","volume":"226","author":"Mamelle","year":"2016","journal-title":"J. Control. Release"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/j.colsurfb.2015.03.018","article-title":"Improved stability and skin permeability of sodium hyaluronate-chitosan multilayered liposomes by Layer-by-Layer electrostatic deposition for quercetin delivery","volume":"129","author":"Jeon","year":"2015","journal-title":"Colloids Surf. B Biointerfaces"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1002\/(SICI)1097-4628(19970103)63:1<125::AID-APP13>3.0.CO;2-4","article-title":"Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers","volume":"63","author":"Calvo","year":"1997","journal-title":"J. Appl. Polym. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1016\/j.ejpb.2013.06.003","article-title":"Hyaluronic acid-coated liposomes for active targeting of gemcitabine","volume":"85","author":"Arpicco","year":"2013","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.ijpharm.2017.10.007","article-title":"Hyalugel-integrated liposomes as a novel ocular nanosized delivery system of fluconazole with promising prolonged effect","volume":"534","author":"Moustafa","year":"2017","journal-title":"Int. J. Pharm."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1144","DOI":"10.3109\/10717544.2014.991952","article-title":"Preparation and ocular pharmacokinetics of hyaluronan acid-modified mucoadhesive liposomes","volume":"23","author":"Lin","year":"2016","journal-title":"Drug Deliv."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/j.ijpharm.2009.02.012","article-title":"Hyaluronic acid coated poly(butyl cyanoacrylate) nanoparticles as anticancer drug carriers","volume":"373","author":"He","year":"2009","journal-title":"Int. J. Pharm."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2351","DOI":"10.1038\/s41598-021-81983-1","article-title":"Development of a novel hyaluronic acid membrane for the treatment of ocular surface diseases","volume":"11","author":"Kim","year":"2021","journal-title":"Sci. Rep."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/12\/2093\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:39:59Z","timestamp":1760168399000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/12\/2093"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,12,5]]},"references-count":45,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2021,12]]}},"alternative-id":["pharmaceutics13122093"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics13122093","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,12,5]]}}}