{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T11:17:56Z","timestamp":1788175076739,"version":"build-2803163510"},"reference-count":48,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2021,1,6]],"date-time":"2021-01-06T00:00:00Z","timestamp":1609891200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Symmetry"],"abstract":"<jats:p>Insulin mucoadhesive buccal films (MBF) are a noninvasive insulin delivery system that offers an advantageous alternative route of administration to subcutaneous injection. One major concern in the formulation of insulin MBF is the preservation of an insulin secondary structure in the presence of the other film components. Buccal films were formulated using chitosan, glycerin, and L-arginine. The MBF-forming solutions (MBF-FS) and the films (MBF) were examined for their chemical and structural stability and for their in vivo activity. Enzyme-Linked Immunosorbent Assay (ELISA) of the insulin-loaded MBF showed that each individualized unit dose was at least loaded with 80% of the insulin theoretical dose. Results of Synchrotron Radiation Circular Dichroism (SRCD) measurements revealed that MBF-FS retained the \u03b1-helices and \u03b2\u2013sheets conformations of insulin. Fourier transform infrared (FTIR)-microspectroscopy (FTIR-MS) examination of insulin MBF revealed the protective action of L-arginine on insulin structure by interacting with chitosan and minimizing the formation of an unordered structure and \u03b2-strand. A blood glucose-lowering effect of insulin MBF was observed in comparison with subcutaneous (S.C) injection using a rat model. As a result; chitosan-based MBFs were formulated and characterized using SRCD and FTIR-MS techniques. Furthermore, the results of in vivo testing suggested the MBFs as a promising delivery system for insulin.<\/jats:p>","DOI":"10.3390\/sym13010088","type":"journal-article","created":{"date-parts":[[2021,1,6]],"date-time":"2021-01-06T20:45:42Z","timestamp":1609965942000},"page":"88","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Characterization of Insulin Mucoadhesive Buccal Films: Spectroscopic Analysis and In Vivo Evaluation"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6559-6592","authenticated-orcid":false,"given":"Maram","family":"Diab","sequence":"first","affiliation":[{"name":"School of Pharmacy, University of Jordan, Amman 11942, Jordan"},{"name":"Al-Taqaddom Pharmaceutical Industries, Amman 11947, Jordan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7872-2084","authenticated-orcid":false,"given":"Al-Sayed","family":"Sallam","sequence":"additional","affiliation":[{"name":"Al-Taqaddom Pharmaceutical Industries, Amman 11947, Jordan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Imad","family":"Hamdan","sequence":"additional","affiliation":[{"name":"School of Pharmacy, University of Jordan, Amman 11942, Jordan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Randa","family":"Mansour","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, Philadelphia University, Amman 19392, Jordan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rohanah","family":"Hussain","sequence":"additional","affiliation":[{"name":"Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot OX11 0DE, Oxfordshire, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4667-6423","authenticated-orcid":false,"given":"Giuliano","family":"Siligardi","sequence":"additional","affiliation":[{"name":"Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot OX11 0DE, Oxfordshire, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9628-6036","authenticated-orcid":false,"given":"Nidal","family":"Qinna","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy and Medical Sciences, Petra University, Amman 11196, Jordan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5878-443X","authenticated-orcid":false,"given":"Enam","family":"Khalil","sequence":"additional","affiliation":[{"name":"School of Pharmacy, University of Jordan, Amman 11942, Jordan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,1,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"S11","DOI":"10.1016\/S1751-9918(10)60004-6","article-title":"Addressing barriers to initiation of insulin in patients with type 2 diabetes","volume":"4","author":"Peyrot","year":"2010","journal-title":"Prim. Care Diabetes"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.jconrel.2009.07.016","article-title":"Orotransmucosal drug delivery systems: A review","volume":"140","author":"Madhav","year":"2009","journal-title":"J. Control. Release"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"254","DOI":"10.3109\/03639040903117348","article-title":"Critical evaluation of permeation enhancers for oral mucosal drug delivery","volume":"36","author":"Sohi","year":"2010","journal-title":"Drug Dev. Ind. Pharm."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1016\/j.ejpb.2008.08.016","article-title":"In Vitro evaluation of natural and methylated cyclodextrins as buccal permeation enhancing system for omeprazole delivery","volume":"71","author":"Figueiras","year":"2009","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1016\/j.ijpharm.2017.01.066","article-title":"Potential of single cationic amino acid molecule \u201cArginine\u201d for stimulating oral absorption of insulin","volume":"521","author":"Kamei","year":"2017","journal-title":"Int. J. Pharm."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4919","DOI":"10.1021\/bi047528r","article-title":"Role of arginine in the stabilization of proteins against aggregation","volume":"44","author":"Baynes","year":"2005","journal-title":"Biochemistry"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1038\/186470a0","article-title":"Symmetry of insulin dimers and hexamers","volume":"186","author":"Low","year":"1960","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1016\/S0022-2836(66)80275-9","article-title":"The Crystal structure of insulin: III. Evidence for a 2-fold axis in rhombohedral zinc insulin","volume":"16","author":"Dodson","year":"1966","journal-title":"J. Mol. Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"492","DOI":"10.2337\/diab.21.2.S492","article-title":"Three-Dimensional atomic structure of insulin and its relationship to activity","volume":"21","author":"Blundell","year":"1972","journal-title":"Diabetes"},{"key":"ref_10","first-page":"434289","article-title":"Inhibitory effects of arginine on the aggregation of bovine insulin","volume":"2012","author":"Varughese","year":"2012","journal-title":"J. Biophys."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Iannuzzi, C., Borriello, M., Portaccio, M., Irace, G., and Sirangelo, I. (2017). Insights into insulin fibril assembly at physiological and acidic pH and related amyloid intrinsic fluorescence. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18122551"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Fasman, G.D. (1996). Circular Dichroism and the Conformational Analysis of Biomolecules 1996, Plenum Press.","DOI":"10.1007\/978-1-4757-2508-7"},{"key":"ref_13","unstructured":"Berova, N., Nakanichi, K., and Woody, R.W. (2000). Circular Dichroism Principles and Applications, Wiley-VCH. [2nd ed.]."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2096","DOI":"10.1002\/prot.25365","article-title":"Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers","volume":"85","author":"Mawhinney","year":"2017","journal-title":"Proteins Struct. Funct. Bioinform."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"7154","DOI":"10.1039\/c0cc02423f","article-title":"Direct observation and pH control of reversed supramolecular chirality in insulin fibrils by vibrational circular dichroism","volume":"46","author":"Kurouski","year":"2010","journal-title":"Chem. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"11863","DOI":"10.1021\/jp3062674","article-title":"On the DMSO-Dissolved State of Insulin: A Vibrational Spectroscopic Study of Structural Disorder","volume":"116","author":"Dzwolak","year":"2012","journal-title":"J. Phys. Chem. B"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5043","DOI":"10.1021\/bi00563a017","article-title":"Conformational dynamics of insulin in solution. Circular dichroic studies","volume":"19","author":"Pocker","year":"1980","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1021\/bi00504a006","article-title":"Estimation of globular protein secondary structure from circular dichroism","volume":"20","author":"Provencher","year":"1981","journal-title":"Biochemistry"},{"key":"ref_19","unstructured":"Erick, M.C., and Yamamoto, H. (2012). Spectroscopic Analysis: Synchrotron Radiation Circular Dichroism. Comprehensive Chirality, Elsevier."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1107\/S0909049511038982","article-title":"Circular dichroism beamline B23 at the Diamond light source","volume":"19","author":"Hussain","year":"2012","journal-title":"J. Syn. Rad."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1412","DOI":"10.1007\/s11051-012-1412-5","article-title":"Gold nanoparticles\u2019 blocking effect on UV-induced damage to human serum albumin","volume":"15","author":"Calzolai","year":"2013","journal-title":"J. Nanopart. Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1002\/bip.360250307","article-title":"Examination of the secondary structure of proteins by deconvolved FTIR spectra","volume":"25","author":"Byler","year":"1986","journal-title":"Biopolymers"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"95","DOI":"10.3109\/10409239509085140","article-title":"The use and misuse of FTIR spectroscopy in the determination of protein structure","volume":"30","author":"Jackson","year":"1995","journal-title":"Crit. Rev. Biochem. Mol. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/0006-291X(83)91016-1","article-title":"Protein structure by Fourier transform infrared spectroscopy: Second derivative spectra","volume":"115","author":"Susi","year":"1983","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/0165-022X(85)90005-3","article-title":"Estimation of B-structure content of proteins by means of deconvolved FTIR spectra","volume":"11","author":"Susi","year":"1985","journal-title":"J. Biochem. Biophys. Methods"},{"key":"ref_26","unstructured":"Mantsch, H.H., and Chapman, D. (1996). Infrared Spectroscopy of Biomolecules, Wiley-Liss."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Delbeck, S., and Heise, H.M. (2020). Quality Assurance of Commercial Insulin Formulations: Novel Assay using Infrared Spectroscopy. J. Diabetes Sci. Technol.","DOI":"10.1177\/1932296820913874"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"952","DOI":"10.1016\/j.ijbiomac.2018.12.192","article-title":"Enzymatically cross-linked arabinoxylan microspheres as oral insulin delivery system","volume":"126","author":"Micard","year":"2019","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1093\/clinchem\/47.3.602","article-title":"Elecsys insulin assay: Free insulin determination and the absence of cross-reactivity with insulin lispro","volume":"47","author":"Sapin","year":"2001","journal-title":"Clin. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"15","DOI":"10.14227\/DT180311P15","article-title":"Simulated biological fluids with possible application in dissolution testing","volume":"18","author":"Marques","year":"2011","journal-title":"Dissolut. Technol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"E3095","DOI":"10.1073\/pnas.1500851112","article-title":"Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy","volume":"112","author":"Micsonai","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Elsayed, A.M., Khaled, A.H., Al Remawi, M.M., Qinna, N.A., Abu Farsakh, H., and Badwan, A.A. (2018). Low molecular weight chitosan-insulin complexes solubilized in a mixture of self-assembled labrosol and plurol oleaque and their glucose reduction activity in rats. Mar. Drugs, 16.","DOI":"10.3390\/md16010032"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Muzaffar, M., and Ahmad, A. (2011). The Mechanism of Enhanced Insulin Amyloid Fibril Formation by NaCl Is Better Explained by a Conformational Change Model. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0027906"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1428","DOI":"10.1016\/j.ijbiomac.2017.10.004","article-title":"Mechanism of co-aggregation in a protein mixture with small additives","volume":"107","author":"Oki","year":"2018","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1016\/j.ijbiomac.2019.09.196","article-title":"Characterization of arginine preventive effect on heat-induced aggregation of insulin","volume":"145","author":"Kurganov","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1301","DOI":"10.1021\/bp0498793","article-title":"Role of arginine in protein refolding, solubilization, and purification","volume":"20","author":"Tsumoto","year":"2004","journal-title":"Biotechnol. Prog."},{"key":"ref_37","first-page":"159","article-title":"Arginine inhibits aggregation of \u03b1-lactalbumin but also decreases its stability: Calorimetric, spectroscopic, and molecular dynamics studies","volume":"78","author":"Kumar","year":"2014","journal-title":"J. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1111\/j.1745-7270.2007.00320.x","article-title":"Fourier transform infrared spectroscopic analysis of protein secondary structures","volume":"39","author":"Kong","year":"2007","journal-title":"Acta Biochim. Biophys. Sin."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1007\/s10973-011-1429-5","article-title":"Physical and chemical characterization insulin-loaded chitosan-TPP nanoparticles","volume":"106","author":"Azevedo","year":"2011","journal-title":"J. Anal. Calorim."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2198","DOI":"10.1007\/s11095-007-9367-4","article-title":"Alginate\/chitosan nanoparticles are effective for oral insulin delivery","volume":"24","author":"Sarmento","year":"2007","journal-title":"Pharm. Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1016\/j.ejpb.2007.11.008","article-title":"Characterization of drug\u2013chitosan interaction by 1 H NMR, FTIR and isothermal titration calorimetry","volume":"69","author":"Boonsongrit","year":"2008","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1002\/masy.201400215","article-title":"Effect of Excipients on the Stability of Insulin Lispro","volume":"351","author":"Misra","year":"2015","journal-title":"Macromol. Symp."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2328","DOI":"10.1016\/j.bbamem.2013.04.012","article-title":"ATR-FTIR: A \u201crejuvenated\u201d tool to investigate amyloid proteins. Biochim","volume":"1828","author":"Sarroukh","year":"2013","journal-title":"Biophys. Acta"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1007\/978-1-4939-2205-5_20","article-title":"Insoluble protein assemblies characterized by fourier transform infrared spectroscopy","volume":"1258","author":"Natalello","year":"2015","journal-title":"Methods Mol. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.1016\/j.bbabio.2007.06.004","article-title":"Infrared spectroscopy of proteins","volume":"1767","author":"Barth","year":"2007","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"382","DOI":"10.1038\/nprot.2015.024","article-title":"Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy","volume":"10","author":"Yang","year":"2015","journal-title":"Nat. Protoc."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"967","DOI":"10.1080\/10837450.2019.1619183","article-title":"Preparation and characterization of insulin chitosan-nanoparticles loaded in buccal films","volume":"24","author":"Alsharif","year":"2019","journal-title":"Pharm. Dev. Technol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.jconrel.2020.07.037","article-title":"Ionic liquid-mediated delivery of insulin to buccal mucosa","volume":"327","author":"Vaidya","year":"2020","journal-title":"J. Control. 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