{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T08:03:02Z","timestamp":1785312182126,"version":"3.55.0"},"reference-count":39,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2020,6,16]],"date-time":"2020-06-16T00:00:00Z","timestamp":1592265600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002341","name":"Academy of Finland","doi-asserted-by":"publisher","award":["311122"],"award-info":[{"award-number":["311122"]}],"id":[{"id":"10.13039\/501100002341","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003125","name":"Suomen Kulttuurirahasto","doi-asserted-by":"publisher","award":["personal grant, LH"],"award-info":[{"award-number":["personal grant, LH"]}],"id":[{"id":"10.13039\/501100003125","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100006306","name":"Sigrid Juselius Foundation","doi-asserted-by":"publisher","award":["n.a."],"award-info":[{"award-number":["n.a."]}],"id":[{"id":"10.13039\/501100006306","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007753","name":"It\u00e4-Suomen Yliopisto","doi-asserted-by":"publisher","award":["Doctoral School of the University of Eastern Finland (S.B.)"],"award-info":[{"award-number":["Doctoral School of the University of Eastern Finland (S.B.)"]}],"id":[{"id":"10.13039\/100007753","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC\/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC\/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology.<\/jats:p>","DOI":"10.3390\/pharmaceutics12060554","type":"journal-article","created":{"date-parts":[[2020,6,16]],"date-time":"2020-06-16T13:20:43Z","timestamp":1592313643000},"page":"554","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":38,"title":["Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6090-1156","authenticated-orcid":false,"given":"Laura","family":"Hellinen","sequence":"first","affiliation":[{"name":"School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70210 Kuopio, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9961-1260","authenticated-orcid":false,"given":"Sina","family":"Bahrpeyma","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70210 Kuopio, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4093-3465","authenticated-orcid":false,"given":"Anna-Kaisa","family":"Rimpel\u00e4","sequence":"additional","affiliation":[{"name":"Drug Research Programme, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marja","family":"Hagstr\u00f6m","sequence":"additional","affiliation":[{"name":"Drug Research Programme, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1224-4331","authenticated-orcid":false,"given":"Mika","family":"Reinisalo","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70210 Kuopio, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arto","family":"Urtti","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70210 Kuopio, Finland"},{"name":"Drug Research Programme, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland"},{"name":"Laboratory of Biohybrid Technologies, Institute of Chemistry, St. Petersburg State University, Peterhoff, St. Petersburg 198504, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,6,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Borovansky, J., and Riley, P.A. (2011). Classical and Nonclassical Melanocytes in Vertebrates. Melanins and Melanosomes: Biosynthesis, Structure, Physiological and Pathological Functions, Wiley-Blackwell.","DOI":"10.1002\/9783527636150.ch12"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1053\/j.seminoncol.2016.02.031","article-title":"Cutaneous Manifestations Associated with Melanoma","volume":"43","author":"Vyas","year":"2016","journal-title":"Semin. Oncol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"13201","DOI":"10.1021\/jp804248h","article-title":"Insights into Melanosomes and Melanin from some Interesting Spatial and Temporal Properties","volume":"112","author":"Simon","year":"2008","journal-title":"J. Phys. Chem. B"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"722","DOI":"10.1111\/j.1365-4362.1979.tb05009.x","article-title":"Patterns and Intensity of Autofluorescence and its Relation to Melanin in Human Epidermis and Hair","volume":"18","author":"Fellner","year":"1979","journal-title":"Int. J. Dermatol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1016\/j.preteyeres.2017.06.005","article-title":"Fluorescence Lifetime Imaging Ophthalmoscopy","volume":"60","author":"Dysli","year":"2017","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1111\/pcmr.12535","article-title":"Fluorescent Quantification of Melanin","volume":"29","author":"Fernandes","year":"2016","journal-title":"Pigment Cell Melanoma Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"26022","DOI":"10.1117\/1.JBO.18.2.026022","article-title":"Use of Multiphoton Tomography and Fluorescence Lifetime Imaging to Investigate Skin Pigmentation in Vivo","volume":"18","author":"Dancik","year":"2013","journal-title":"J. Biomed. Opt."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.addr.2017.12.008","article-title":"Implications of Melanin Binding in Ocular Drug Delivery","volume":"126","author":"Rimpela","year":"2018","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1016\/j.ejps.2017.07.027","article-title":"Melanin Binding Study of Clinical Drugs with Cassette Dosing and Rapid Equilibrium Dialysis Inserts","volume":"109","author":"Pelkonen","year":"2017","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1167\/iovs.05-0945","article-title":"Concentrations of Betaxolol in Ocular Tissues of Patients with Glaucoma and Normal Monkeys After 1 Month of Topical Ocular Administration","volume":"47","author":"Hollo","year":"2006","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2977","DOI":"10.1021\/acs.molpharmaceut.5b00787","article-title":"Drug Distribution to Retinal Pigment Epithelium: Studies on Melanin Binding, Cellular Kinetics, and SPECT\/CT Imaging","volume":"13","author":"Rimpela","year":"2016","journal-title":"Mol. Pharm."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/0378-5173(84)90103-0","article-title":"Effect of Ocular Pigmentation on Pilocarpine Pharmacology in the Rabbit Eye. I. Drag Distribution and Metabolism","volume":"18","author":"Salminen","year":"1984","journal-title":"Int. J. Pharm."},{"key":"ref_13","first-page":"79","article-title":"Iris Pigmentation and Atropine Mydriasis","volume":"197","author":"Salazar","year":"1976","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_14","first-page":"671","article-title":"An Explanation for the Long Duration of Mydriatic Effect of Atropine in Eye","volume":"15","author":"Salazar","year":"1976","journal-title":"Investig. Ophthalmol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/0378-5173(84)90132-7","article-title":"Effect of Ocular Pigmentation on Pilocarpine Pharmacology in the Rabbit Eye. II. Drug Response","volume":"19","author":"Urtti","year":"1984","journal-title":"Int. J. Pharm."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1016\/j.jconrel.2018.05.034","article-title":"Melanin Targeting for Intracellular Drug Delivery: Quantification of Bound and Free Drug in Retinal Pigment Epithelial Cells","volume":"283","author":"Rimpela","year":"2018","journal-title":"J. Control. Release"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4890","DOI":"10.1021\/acs.molpharmaceut.9b00769","article-title":"Establishment of an in Vitro-in Vivo Correlation for Melanin Binding and the Extension of the Ocular Half-Life of Small-Molecule Drugs","volume":"12","author":"Jakubiak","year":"2019","journal-title":"Mol. Pharm."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1490","DOI":"10.1167\/iovs.12-10169","article-title":"Assessing a Novel Depot Delivery Strategy for Noninvasive Administration of VEGF\/PDGF RTK Inhibitors for Ocular Neovascular Disease","volume":"54","author":"Robbie","year":"2013","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1183","DOI":"10.3109\/03602538409033561","article-title":"The Melanin Binding of Drugs and its Implications","volume":"15","author":"Ings","year":"1984","journal-title":"Drug Metab. Rev."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1006\/rtph.1998.1243","article-title":"Binding of Drugs to Eye Melanin is Not Predictive of Ocular Toxicity","volume":"28","author":"Leblanc","year":"1998","journal-title":"Regul. Toxicol. Pharmacol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1186\/s13046-018-0983-0","article-title":"Targeted Radiotherapy of Pigmented Melanoma with (131)I-5-IPN","volume":"37","author":"Xu","year":"2018","journal-title":"J. Exp. Clin. Cancer Res."},{"key":"ref_22","first-page":"1","article-title":"Neuromelanin Detection by Magnetic Resonance Imaging (MRI) and its Promise as a Biomarker for Parkinson\u2019s Disease","volume":"4","author":"Sulzer","year":"2018","journal-title":"NPJ Parkinson\u2019s Dis."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Solano, F. (2017). Melanin and Melanin-Related Polymers as Materials with Biomedical and Biotechnological Applications-Cuttlefish Ink and Mussel Foot Proteins as Inspired Biomolecules. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18071561"},{"key":"ref_24","first-page":"405","article-title":"The Reaction of Uveal Pigment in Vitro with Polycyclic Compounds","volume":"3","author":"POTTS","year":"1964","journal-title":"Investig. Ophthalmol."},{"key":"ref_25","first-page":"517","article-title":"Uveal Pigment and Phenothiazine Compounds. Trans","volume":"60","author":"POTTS","year":"1962","journal-title":"Am. Ophthalmol. Soc."},{"key":"ref_26","first-page":"399","article-title":"Further Studies Concerning the Accumulation of Polycyclic Compounds on Uveal Melanin","volume":"3","author":"POTTS","year":"1964","journal-title":"Investig. Ophthalmol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"10106","DOI":"10.1021\/acs.jmedchem.8b01281","article-title":"Understanding Molecular Drivers of Melanin Binding to Support Rational Design of Small Molecule Ophthalmic Drugs","volume":"61","author":"Jakubiak","year":"2018","journal-title":"J. Med. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.molstruc.2014.03.009","article-title":"MicroScale Thermophoresis: Interaction Analysis and Beyond","volume":"1077","author":"Wanner","year":"2014","journal-title":"J. Mol. Struct."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/j.ymeth.2012.12.005","article-title":"Microscale Thermophoresis Quantifies Biomolecular Interactions Under Previously Challenging Conditions","volume":"59","author":"Seidel","year":"2013","journal-title":"Methods"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"15185","DOI":"10.1021\/ja505412p","article-title":"Transferring Biomarker into Molecular Probe: Melanin Nanoparticle as a Naturally Active Platform for Multimodality Imaging","volume":"136","author":"Fan","year":"2014","journal-title":"J. Am. Chem. Soc."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1251","DOI":"10.1021\/acs.molpharmaceut.5b00783","article-title":"Interpretation of Ocular Melanin Drug Binding Assays. Alternatives to the Model of Multiple Classes of Independent Sites","volume":"13","author":"Manzanares","year":"2016","journal-title":"Mol. Pharm."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2467","DOI":"10.1021\/mp4000822","article-title":"Rapid Measurement of Intracellular Unbound Drug Concentrations","volume":"10","author":"Mateus","year":"2013","journal-title":"Mol. Pharm."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1089\/jop.1992.8.35","article-title":"Quantitative Determination of the Melanin Contents in Ocular Tissues from Human Blue and Brown Eyes","volume":"8","author":"Menon","year":"1992","journal-title":"J. Ocul. Pharmacol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1111\/j.1751-1097.2008.00316.x","article-title":"Role of Ocular Melanin in Ophthalmic Physiology and Pathology","volume":"84","author":"Hu","year":"2008","journal-title":"Photochem. Photobiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1034\/j.1600-0749.2003.00072.x","article-title":"Quantitative Analysis of Eumelanin and Pheomelanin in Humans, Mice, and Other Animals: A Comparative Review","volume":"16","author":"Ito","year":"2003","journal-title":"Pigment Cell Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2549","DOI":"10.1021\/acs.molpharmaceut.9b00157","article-title":"Influence of Melanin Characteristics on Drug Binding Properties","volume":"16","author":"Jakubiak","year":"2019","journal-title":"Mol. Pharm."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1016\/0076-6879(74)31043-9","article-title":"Isolation of Melanin Granules","volume":"31","author":"Menon","year":"1974","journal-title":"Methods Enzymol."},{"key":"ref_38","first-page":"32","article-title":"Binding of Antiglaucomatous Drugs to Synthetic Melanin and their Hypotensive Effects on Pigmented and Nonpigmented Rabbit Eyes","volume":"37","author":"Nagata","year":"1993","journal-title":"Jpn. J. Ophthalmol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"13761","DOI":"10.1038\/s41598-019-50324-8","article-title":"Characterization of Artificially Re-Pigmented ARPE-19 Retinal Pigment Epithelial Cell Model","volume":"9","author":"Hellinen","year":"2019","journal-title":"Sci. Rep."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/12\/6\/554\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:39:24Z","timestamp":1760175564000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/12\/6\/554"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,6,16]]},"references-count":39,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2020,6]]}},"alternative-id":["pharmaceutics12060554"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics12060554","relation":{},"ISSN":["1999-4923"],"issn-type":[{"value":"1999-4923","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,6,16]]}}}