RESSALVA Atendendo solicitação da autora, o texto completo desta Tese será disponibilizado somente a partir de 17/07/2025. O OH HO 3 3 OH H COOCH O OH HO 3 3 OH H COOCH A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 Nano-in-nano containing curcumin and benzydamine hydrochloride for the treatment of Vulvovaginal Candidiasis: Nano-in-nano containing curcumin and benzydamine hydrochloride for the treatment of Vulvovaginal Candidiasis: from development to biological application in vitro and in vivo from development to biological application in vitro and in vivo N an o- in -n an o co n ta in in g c u rc u m in a n d b en zy d am in e h yd ro ch lo ri d e fo r th e tr ea tm en t of V u lv ov ag in al C an d id ia si s Nano-in-nano containing curcumin and benzydamine hydrochloride for the treatment of vulvovaginal candidiasis: from development to biological application in vitro and in vivo Gabriela Corrêa Carvalho Copyright © 2024 by Gabriela Corrêa Carvalho Cover: Gabriela Corrêa Carvalho, Maria Clara Ribeiro Maciel and Dr. Ana Silvia Sartori Barraviera Seabra Ferreira Printed by Gilderprint ISBN: 978-94-6496-148-5 Copyright © 2024 by Gabriela Corrêa Carvalho Cover: Gabriela Corrêa Carvalho, Maria Clara Ribeiro Maciel and Dr. Ana Silvia Sartori Barraviera Seabra Ferreira Printed by Gilderprint ISBN: 978-94-6496-148-5 Nano-in-nano containing curcumin and benzydamine hydrochloride for the treatment of vulvovaginal candidiasis: from development to biological application in vitro and in vivo PhD thesis to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. J.M.A. Scherpen and in accordance with the decision by the College of Deans and to obtain the degree of PhD at the São Paulo State University on the authority of the Rector Magnificus Prof. P. Barretti and in accordance with the decision by the Pro-Rectory of Post-Graduation Double PhD degree This thesis will be defended in public on Wednesday 17 July 2024 at 14:30 hours by Gabriela Corrêa Carvalho born on 3 May 1994 in Botucatu, Brazil Carvalho, Gabriela Corrêa. C331n Nano-in-nano containing curcumin and benzydamine hydrochloride for the treatment of vulvovaginal candidiasis: from development to biological application in vitro and in vivo = Nano-in-nano contendo curcumina e cloridrato de benzidamina para tratamento de Candidíase Vulvovaginal: do desenvolvimento à aplicação biológica in vitro e in vivo / Gabriela Corrêa Carvalho. – Groningen; Araraquara, 2024. 468 f. : il. Thesis (Doctorate with double degree) – Universidade Estadual Paulista “Júlio de Mesquita Filho”. Faculdade de Ciências Farmacêuticas. Programa de Pós- Graduação em Ciências Farmacêuticas – University Medical Center Groningen. NANOBIOMED: Nanotechnology & Biophysics in Medicine Program. Advisor: Marlus Chorilli (UNESP). Advisor: Hélder Almeida Santos (UMCG). Co-advisor: Taís Maria Bauab (UNESP). Co-advisor: Brandon W. Peterson (UMCG). Co-advisor: Rafael Miguel Sábio (UNESP). 1. Nanoparticles. 2. Candidiasis - Vulvovaginal. 3. Curcumin. 4. Drug resistance - Fungal. 5. Hydrogels. I. Chorilli, Marlus, orient. II. Santos, Hélder Almeida, orient. III. Bauab, Taís Maria, coorient. IV. Peterson, Brandon W., coorient. V. Sábio, Rafael Miguel, coorient. VI. Title. Diretoria do Serviço Técnico de Biblioteca e Documentação - Faculdade de Ciências Farmacêuticas UNESP - Campus de Araraquara CAPES: 33004030078P6 The Cataloging in Publication data cannot be altered Câmpus de Araraquara F a c u l d a d e d e C i ê n c i a s F a r m a c ê u t i c a s – S e ç ã o T é c n i c a d e P ó s - g r a d u a ç ã o R o d o v i a A r a r a q u a r a - J a ú , K m 1 - C E P 1 4 8 0 0 - 9 0 3 - A r a r a q u a r a - S P . - B r a s i l t e l 1 6 3 3 0 1 - 6 9 0 0 – 6 9 0 1 – p g . f c f @ u n e s p . b r CERTIFICADO DE APROVAÇÃO TÍTULO DA TESE: Nano-in-nano containing curcumin and benzydamine hydrochloride for the treatment of vulvovaginal candidiasis: from development to biological application in vitro and in vivo AUTORA: GABRIELA CORREA CARVALHO ORIENTADOR: MARLUS CHORILLI ORIENTADOR: HÉLDER ALMEIDA SANTOS COORIENTADOR: RAFAEL MIGUEL SÁBIO COORIENTADORA: TAIS MARIA BAUAB COORIENTADOR: BRANDON W PETERSON Aprovada como parte das exigências para obtenção do Título de Doutora em Ciências Farmacêuticas, área: Pesquisa e Desenvolvimento de Fármacos e Medicamentos, em regime de Cotutela entre a Universidade Estadual Paulista “Julio de Mesquita Filho” e a University of Groningen, Netherlands. Comissão Examinadora: Prof. Dr. MARLUS CHORILLI (Participação Virtual) Departamento de Farmacos e Medicamentos / Faculdade de Ciencias Farmaceuticas do Campus de Araraquara da Unesp Prof. Dr. HÉLDER ALMEIDA SANTOS (Participação Presencial) Department of Biomedical Engineering / University Medical Center Groningen - University of Groningen (Netherlands) Prof. Dr. BRUNO FILIPE CARMELINO CARDOSO SARMENTO (Participação Virtual) Instituto de Investigação e Inovação em Saúde (i3S) / Universidade do Porto (Portugal) Prof. Dr. CHRISTIAN CELIA (Participação Presencial) Dipartimento di Farmacia / Università degli Studi G. d'Annunzio Chieti (Itália) Profa. Dra. TEREZINHA DE JESUS ANDREOLI PINTO (Participação Virtual) Câmpus de Araraquara F a c u l d a d e d e C i ê n c i a s F a r m a c ê u t i c a s – S e ç ã o T é c n i c a d e P ó s - g r a d u a ç ã o R o d o v i a A r a r a q u a r a - J a ú , K m 1 - C E P 1 4 8 0 0 - 9 0 3 - A r a r a q u a r a - S P . - B r a s i l t e l 1 6 3 3 0 1 - 6 9 0 0 – 6 9 0 1 – p g . f c f @ u n e s p . b r Departamento de Farmácia / Faculdade de Ciências Farmacêuticas da Universidade de São Paulo (USP) Profa. Dra. INGE ZUHORN (Participação Presencial) Department of Biomedical Engineering / University Medical Center Groningen - University of Groningen (Netherlands) Prof. Dr. JAN MAARTEN VAN DIJL (Participação Presencial) Department of Medical Microbiology / University Medical Center Groningen - University of Groningen (Netherlands) Profa.Dra. SANDRA HELENA PULCINELLI (Participação Virtual) Departamento de Química Analítica, Físico-Química e Inorgânica / Instituto de Química do Campus de Araraquara da Unesp Araraquara, 17 de julho de 2024 Supervisors Prof. H. Almeida Santos Prof. M. Chorilli Co-supervisors Dr. B.W. Peterson Prof. T.M. Bauab Daily supervisor Dr. R.M. Sábio Assessment Committee Prof. I. Zuhorn Prof. J.M. van Dijl Prof. S.H. Pulcinelli Prof. T.J. Andreoli Pinto Gésinda Geertsema-Doornbusch Jiachen Li Paranymphs Gésinda Geertsema-Doornbusch Jiachen Li I dedicate this work to Prof.Ione Corrêa, my mom, for always being an example for me, love you! Table of contents ABBREVIATIONS AND SYMBOLS LIST 2 CHAPTER 1 Introduction and objective of the thesis 11 CHAPTER 2 Prevalence of vulvovaginal candidiasis in Brazil: A systematic review 21 CHAPTER 3 Infection caused by Candida auris: State of the art 45 CHAPTER 4 Lycopene: From tomato to its nutraceutical use and its association with nanotechnology 99 CHAPTER 5 An overview of properties and analytical methods for lycopene in organic nanocarriers 129 CHAPTER 6 Highlights in mesoporous silica nanoparticles as a multifunctional controlled drug delivery nanoplatform for infectious diseases treatment 155 CHAPTER 7 Physicochemical characterization of a lycopene-loaded mesoporous silica nanoparticle formulation 215 CHAPTER 8 Lycopene, mesoporous silica nanoparticles and their Association: a possible alternative against vulvovaginal candidiasis? 249 CHAPTER 9 Cetyltrimethylammonium bromide in the synthesis of mesoporous silica nanoparticles: general aspects and in vitro toxicity 269 CHAPTER 10 From curcuma longa to its nutraceutic use and its association with nanotechnology 307 CHAPTER 11 Nano-in-nano particles dual-loaded with curcumin and benzydamine hydrochloride for the treatment of vulvovaginal candidiasis: From development to biological application in vitro and in vivo 365 CHAPTER 12 Highlights in poloxamer-based drug delivery systems as strategy at local application for vaginal infections 403 CHAPTER 13: General discussion 435 CHAPTER 14 Conclusions and future perspectives 441 ABSTRACT 443 RESUMO 444 SAMENVATTING 445 APPENDIX 1 447 ACKNOWLEDGMENTS 451 ABOUT THE AUTHOR 455 Table of contents ABBREVIATIONS AND SYMBOLS LIST 2 CHAPTER 1 Introduction and objective of the thesis 11 CHAPTER 2 Prevalence of vulvovaginal candidiasis in Brazil: A systematic review 21 CHAPTER 3 Infection caused by Candida auris: State of the art 45 CHAPTER 4 Lycopene: From tomato to its nutraceutical use and its association with nanotechnology 99 CHAPTER 5 An overview of properties and analytical methods for lycopene in organic nanocarriers 129 CHAPTER 6 Highlights in mesoporous silica nanoparticles as a multifunctional controlled drug delivery nanoplatform for infectious diseases treatment 155 CHAPTER 7 Physicochemical characterization of a lycopene-loaded mesoporous silica nanoparticle formulation 215 CHAPTER 8 Lycopene, mesoporous silica nanoparticles and their Association: a possible alternative against vulvovaginal candidiasis? 249 CHAPTER 9 Cetyltrimethylammonium bromide in the synthesis of mesoporous silica nanoparticles: general aspects and in vitro toxicity 269 CHAPTER 10 From curcuma longa to its nutraceutic use and its association with nanotechnology 307 CHAPTER 11 Nano-in-nano particles dual-loaded with curcumin and benzydamine hydrochloride for the treatment of vulvovaginal candidiasis: From development to biological application in vitro and in vivo 365 CHAPTER 12 Highlights in poloxamer-based drug delivery systems as strategy at local application for vaginal infections 403 CHAPTER 13: General discussion 435 CHAPTER 14 Conclusions and future perspectives 441 ABSTRACT 443 RESUMO 444 SAMENVATTING 445 APPENDIX 1 447 ACKNOWLEDGMENTS 451 ABOUT THE AUTHOR 455 1 Abbreviations and symbols list % Percentage ~ Approximately ± Plus–minus ∆mMSN Empty silica mass variation ∆mS Sample mass variation ∆mT Total mass variation ° Degree µg Microgram µL Microliter µm Micrometre µM Micromolar µmol Micromole 10T1/2 Clonal cell line 1O2 Singlet oxygen 2D Two dimensional 3CL 3-chymotrypsin-like 3D Three dimensional 99mTc Technetium-99m A549 Human lung cancer cell line Ab Antibody ABC Adenosine triphosphate binding cassette ABCA1 Adenosine triphosphate -binding cassette 1 ACV Acyclovir AFLP Amplified fragment length polymorphism AgNPs Silver nanoparticles AIBA Sodium dihydrochloride 2,2'- Azobis (2-methylproprylnamide) AIDS Acquired immunodeficiency syndrome AKT Protein kinase B AL Alagoas State ALT Alanine aminotransferase ANOVA Analysis of variance AP Acetophenone APP/PS1 Transgenic mice that overexpress human mutant amyloid precursor protein and presenilin -1 APTES 3-Aminopropyltriethoxysilane ARE Antioxidant response element ARs Aspect ratios AST Aspartate transaminase ATCC American Type Culture Collection ATP Adenosine triphosphate ATR Attenuated total reflectance AuNPs Gold nanoparticles AuNTs Gold nanotubes BA Bahia State BAL Bronchoalveolar lavage BALB A portmanteau of "Bagg" and "Albino" Bax B-cell lymphoma 2-associated X-protein BCID Blood Culture Identification Bcl-2 B-cell lymphoma 2 Bcl-xL B-cell lymphoma-extra large BDNF Brain-derived neurotrophic factor BES Benzenesulfonyl 2 1 Abbreviations and symbols list % Percentage ~ Approximately ± Plus–minus ∆mMSN Empty silica mass variation ∆mS Sample mass variation ∆mT Total mass variation ° Degree µg Microgram µL Microliter µm Micrometre µM Micromolar µmol Micromole 10T1/2 Clonal cell line 1O2 Singlet oxygen 2D Two dimensional 3CL 3-chymotrypsin-like 3D Three dimensional 99mTc Technetium-99m A549 Human lung cancer cell line Ab Antibody ABC Adenosine triphosphate binding cassette ABCA1 Adenosine triphosphate -binding cassette 1 ACV Acyclovir AFLP Amplified fragment length polymorphism AgNPs Silver nanoparticles AIBA Sodium dihydrochloride 2,2'- Azobis (2-methylproprylnamide) AIDS Acquired immunodeficiency syndrome AKT Protein kinase B AL Alagoas State ALT Alanine aminotransferase ANOVA Analysis of variance AP Acetophenone APP/PS1 Transgenic mice that overexpress human mutant amyloid precursor protein and presenilin -1 APTES 3-Aminopropyltriethoxysilane ARE Antioxidant response element ARs Aspect ratios AST Aspartate transaminase ATCC American Type Culture Collection ATP Adenosine triphosphate ATR Attenuated total reflectance AuNPs Gold nanoparticles AuNTs Gold nanotubes BA Bahia State BAL Bronchoalveolar lavage BALB A portmanteau of "Bagg" and "Albino" Bax B-cell lymphoma 2-associated X-protein BCID Blood Culture Identification Bcl-2 B-cell lymphoma 2 Bcl-xL B-cell lymphoma-extra large BDNF Brain-derived neurotrophic factor BES Benzenesulfonyl 2 BET Brunauer–Emmett–Teller BHK-21 Baby Hamster Kidney cell line BLT Bone marrow, liver, thymus - humanized mice bMECs Bovine mammary epithelial cells BMSCs Bone mesenchymal stem cells BNZ Benzydamine hydrochloride BNZ@NIN Benzydamine hydrochloride loaded in the nano-in-nano BV Bacterial vaginosis BZ Benznidazole C14TAB Tetradecyltrimethylammonium bromide ca. Approximately CaCo-2 Cell line derived from a colorectal adenocarcinoma patient Caco-2/TC7 A Caco-2 cell line subclones CaSki Cervical cancer cell line CAT Catalase CCL-119 Human acute lymphoblastic leucemia cell line CD-1 Outbred mice derived from outbred Swiss mice developed at the Anti- Cancer Center in Lausanne, Switzerland CDC Center of Disease Control and Prevention CDR1 Candida drug resistance 1 CE Ceará State CFU Colony forming units CFZ Clofazimine CG-MS Gas chromatography/Mass spectrometry CI Confidence interval CINAHL Cumulative Index to Nursing, and Allied Health Literature CIP Ciprofloxacin CLSI Clinical and Laboratory Standards Institute cm-1 Wavenumber cm2 Square centimetre cm3 Cubic centimetre CMC Critical micelle concentration CMT critical micellar temperature COK-12 Centrum Voor Oppervlaktechemie en Katalyse/Centre for Research Chemistry and Catalysis ConA Concanavalin A COVID 19 Coronavirus disease 2019 CP/MAS Cross-polarization/ Magic-Angle-Spinning CQDs Carbon quantum-dots cRGDY Cyclic arginine-glycine-aspartic acid-tyrosine CRL-1490 Human lung fibroblast cell line CRL-1596 Human Burkitt's lymphoma cell line CryoTEM Transmission electron cryomicroscopy CS Chitosan succinate CT-26 Mouse colon carcinoma cell line CTAB Cetyltrimethylammonium bromide CUR Curcumin CUR@MSN Curcumin incorporated in mesoporous silica nanoparticle CUR+BNZ@HG Curcumin and benzydamine dispersed in hydrogel CUR+BNZ@NIN Curcumin and benzydamine hydrochloride dual loaded in the nano-in- nano CUR+BNZ@NIN@HG Curcumin and benzydamine hydrochloride dual loaded in the nano-in- nano dispersed in hydrogel CVC Central venous catheter Da Dalton 2 3 3 DAD Diode array detector DAMO [3-(2-aminoethylamino) propyl]trimethoxysilane DC Dendritic cells DFO Desferrioxamine DHA Drug: H + antiporter dL Deciliter DL Detection limit DLS Dynamic light scattering DMEM Dulbecco’s modified Eagle medium DMPC 1,2-Dimyristoyl-sn-glycero-3-phosphocholine DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DNase Deoxyribonuclease DOXY Doxycycline DPPH 2,2-Diphenyl-1-picrylhydrazyl DSC Differential scanning calorimetry DSPC 1,2-Distearoyl-sn-glycero-3-phosphocholine DTPA Diethylenetriaminepentaacetic acid DW Dry weight EA Ethyl acetate EAE Enzyme-assisted extraction EC Ethyl cellulose ECDC European Center for Disease Prevention and Control ECO Econazole EDTA Trypsin-ethylenediaminetetraacetic acid EDX Energy dispersive X-ray EE Encapsulation efficiency EMA European Medicines Agency End1/E6E7 Human endometriosis cell line EPS Extracellular polymeric matrix ERG11 Lanosterol 14 α-demethylase gene ERK Extracellular Signal-Regulated Kinase ES Espírito Santo State ETGFA Expansible thermal gelling foam aerosol ETH Ethionamide EUCAST European Committee on Antimicrobial Susceptibility Testing F Freeze f Flake FA Formic acid FB11 Francisella tularensis LPS antibody FBS Fetal bovine serum FDA Food and Drug Administration FICI Fractional inhibitory concentration index FKS1 1.3-Beta-glucan synthase gene FL Fluorescein FMB-01 Clinical strain resistant to azoles provided by the Medical School of São Paulo State University FP Fungal pathogen FSM Folded sheets of mesoporous materials FTIR Fourier-transform infrared spectroscopy FtsZ Filamenting temperature-sensitive mutant Z g Gram GAGs Glycosaminoglycans GEN Gentamicin GFP Green fluorescent protein 4 3 DAD Diode array detector DAMO [3-(2-aminoethylamino) propyl]trimethoxysilane DC Dendritic cells DFO Desferrioxamine DHA Drug: H + antiporter dL Deciliter DL Detection limit DLS Dynamic light scattering DMEM Dulbecco’s modified Eagle medium DMPC 1,2-Dimyristoyl-sn-glycero-3-phosphocholine DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DNase Deoxyribonuclease DOXY Doxycycline DPPH 2,2-Diphenyl-1-picrylhydrazyl DSC Differential scanning calorimetry DSPC 1,2-Distearoyl-sn-glycero-3-phosphocholine DTPA Diethylenetriaminepentaacetic acid DW Dry weight EA Ethyl acetate EAE Enzyme-assisted extraction EC Ethyl cellulose ECDC European Center for Disease Prevention and Control ECO Econazole EDTA Trypsin-ethylenediaminetetraacetic acid EDX Energy dispersive X-ray EE Encapsulation efficiency EMA European Medicines Agency End1/E6E7 Human endometriosis cell line EPS Extracellular polymeric matrix ERG11 Lanosterol 14 α-demethylase gene ERK Extracellular Signal-Regulated Kinase ES Espírito Santo State ETGFA Expansible thermal gelling foam aerosol ETH Ethionamide EUCAST European Committee on Antimicrobial Susceptibility Testing F Freeze f Flake FA Formic acid FB11 Francisella tularensis LPS antibody FBS Fetal bovine serum FDA Food and Drug Administration FICI Fractional inhibitory concentration index FKS1 1.3-Beta-glucan synthase gene FL Fluorescein FMB-01 Clinical strain resistant to azoles provided by the Medical School of São Paulo State University FP Fungal pathogen FSM Folded sheets of mesoporous materials FTIR Fourier-transform infrared spectroscopy FtsZ Filamenting temperature-sensitive mutant Z g Gram GAGs Glycosaminoglycans GEN Gentamicin GFP Green fluorescent protein 4 GPI Glycophosphatidylinositol or glycosylphosphatidylinositol GPTMS (3-Glycidoxypropy) trimethoxysilane GQDs Graphene quantum-dots GSH-Px Glutathione peroxidase GST Recombinant Hepatitis E Virus ORF2 Protein Gwt1 Fungal enzyme inositol acylase h Hour H9c2 Cardiac myofibroblast cells HA Hydroxyapatite HaCaT Human epidermal keratinocyte cell line HDL High-density lipoprotein HEC-1-A Human Endometrial cancer cell line HEK-293T Human embryonic kidney 293 cells HeLa Human epithelial cell Hep-2 Human larynx epithelioma cancer cell line HEPES buffer 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid buffer HepG2 Human liver hepatocellular carcinoma cell line HIV Human immunodeficiency virus HLB Hydrophilic-lipophilic balance HMG-CoA 3-Hydroxy-3-methylglutaryl coenzyme A HMM-33 Hiroshima Mesoporous Material-33 HMSNs Hollow MSNs HO-1 Heme oxidase HOMSNs Hollow oblate mesoporous silica nanoparticle HOS Human osteosarcoma HPLC High-performance liquid chromatography HPMC Hydroxypropyl Methylcellulose HPV Human papillomavirus HR-TEM High-resolution transmission electron microscopy HSP Heat shock protein HSV Herpes simplex virus HT-29 and HT29/MTX Human colon carcinoma cell line HTLV Human T cell lymphotropic virus Hyal Hyaluronidase i.d Internal Diameter IC50 Half-maximal inhibitory concentration ICR Institute of Cancer Research ICU Intensive Care Unit IE Impregnation efficiency IFN Interferons IGF-1R Insulin-like growth factor 1 IgG Immunoglobulin G IL Interleukin INH Isoniazid IP Intraperitoneally IR Infrared ITS Internal transcribed spacer IUDs Intrauterine Devices IUPAC International Union of Pure and Applied Chemistry IV Intravenous J Joule JAK Janus kinase JNK c-Jun NH2-terminal kinase K Retention factor kcal Kilocalories 4 5 5 KeV Kilo Electron Volts kg Kilogram kHz Kilohertz kJ Kilojoule km-1 Kilometre power-1 km2 Square kilometre L Liter L Liquid L0-2 Human hepatocyte cell line LAMP loop-mediated isothermal amplification LC Loading (or load) capacity LC-MSNs Lipid-coated mesoporous silica nanoparticles LD Loading degree LD50 Lethal dose 50 LDH Lactate dehydrogenase LDL Low-density lipoprotein LED Light Emitting Diode LEVO Levofloxacin LILACS Latin American and Caribbean Literature in Health Sciences Log Logarithm LP Liposomes LPS Lipopolisaccharide LU Ubiquicidin onto lipid bilayer LVS Live vaccine strain LXRα Liver X receptor α LYC Lycopene LYC@MSN Mesoporous silica nanoparticle incorporated with lycopene LYZ Lysozyme M Mitotic phase M Moles per liter m2 Square meter m3 Cubic metre MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight MAPK Mitogen-activated protein kinase MBI 1-Methyl-1H-benzimidazole MCF-7 Human breast adenocarcinoma cell line MCM Mobil Crystalline Materials or Mobil Composition of Matter MDA Malondialdehyde MDCK Madin-Darby Canine Kidney cells MFC Minimum fungicide concentration MFS Major Facilitator Superfamily MFX Moxifloxacin mg Milligram MG-63 Human osteoblast cell line MGCE Magnetic glassy carbon electrode MHB Mueller Hinton Broth MIC Minimum Inhibitory Concentration min Minutes miR MicroRNA mL Millilitre ML-336 A quinazolinone-based compound mM Millimolar mm Millimeter mm3 Cubic millimetre MMC Minimum microbicide concentration 6 5 KeV Kilo Electron Volts kg Kilogram kHz Kilohertz kJ Kilojoule km-1 Kilometre power-1 km2 Square kilometre L Liter L Liquid L0-2 Human hepatocyte cell line LAMP loop-mediated isothermal amplification LC Loading (or load) capacity LC-MSNs Lipid-coated mesoporous silica nanoparticles LD Loading degree LD50 Lethal dose 50 LDH Lactate dehydrogenase LDL Low-density lipoprotein LED Light Emitting Diode LEVO Levofloxacin LILACS Latin American and Caribbean Literature in Health Sciences Log Logarithm LP Liposomes LPS Lipopolisaccharide LU Ubiquicidin onto lipid bilayer LVS Live vaccine strain LXRα Liver X receptor α LYC Lycopene LYC@MSN Mesoporous silica nanoparticle incorporated with lycopene LYZ Lysozyme M Mitotic phase M Moles per liter m2 Square meter m3 Cubic metre MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight MAPK Mitogen-activated protein kinase MBI 1-Methyl-1H-benzimidazole MCF-7 Human breast adenocarcinoma cell line MCM Mobil Crystalline Materials or Mobil Composition of Matter MDA Malondialdehyde MDCK Madin-Darby Canine Kidney cells MFC Minimum fungicide concentration MFS Major Facilitator Superfamily MFX Moxifloxacin mg Milligram MG-63 Human osteoblast cell line MGCE Magnetic glassy carbon electrode MHB Mueller Hinton Broth MIC Minimum Inhibitory Concentration min Minutes miR MicroRNA mL Millilitre ML-336 A quinazolinone-based compound mM Millimolar mm Millimeter mm3 Cubic millimetre MMC Minimum microbicide concentration 6 mmol Millimole MMP Matrix metalloproteinase MNZ Metronidazole mol Mole MONPs Metal oxide nanoparticles MPA Multiplex Probe Amplification MPO Myeloperoxidase MPP+ 1-Methyl-4-phenylpyridinium mRNA Messenger ribonucleic acid MRSA Methicillin-resistant S. Aureus MSN Mesoporous silica nanoparticles MSN-C Calcined mesoporous silica nanoparticle MSN-R Refluxed mesoporous silica nanoparticle MSTO Human mesothelioma cell line MTBE Methyl tert-butyl ether mTOR Mammalian target of rapamycin MTT Thiazolyl Blue Tetrazolium romide MTZ Metronidazole mV Millivolt mW Milliwatt MXF Moxifloxacin n Total number of observations or population size n.a. Not available N3 3-[2-(2-Aminoethylamino)ethylamino]propyltrimethoxysilane NA No activity in the tested range NaDC Sodium deoxycholate NADH Nicotinamide adenine dinucleotide hydrogen NADPH Nicotinamide adenine dinucleotide phosphate hydrogen NE Nanoemulsions NET Trapping extracellular neutrophils NFE2L2 Nuclear factor E2-related factor 2 NF-kB Nuclear factor kappa B NFX Norfloxacin ng Nanogram NHDF Normal Human Dermal Fibroblasts NI Niosomes NIH/3T3 Mouse fibroblast cell line NIN Nano-in-nano NIR Near-infrared NLC Nanostructured lipid carriers nm Nanometer nM Nanomolar NMR Nuclear Magnetic Resonance NPs Nanoparticles o Oven O/W Oil-in-Water ºC Celsius ORF2 Open reading frames P Paste p/p0 Equilibrium pressure divided by the saturation pressure PA Pará State PAMPs Pathogen-associated molecular patterns Panc1 Human pancreatic ductal adenocarcinoma cell line PAS Periodic acid-Schiff PBMCs Peripheral blood mononuclear cells 6 7 7 PBS Phosphate-buffered saline PCR Polymerase chain reaction PCV2 Porcine circovirus type 2 PDA Photodiode arrays PDI Polydispersity index PDMAEMA Poly(2-(dimethylamino)ethyl methacrylate) PDT Photodynamic therapy PEG Polyethylene glycol PEI Poly(ethylene imine) PEO Poly(ethylene oxide) PI3K Phosphoinositide 3-kinase PICO Population; intervention; comparation and outcome PK15 Pig kidney cell line PLGA Poly(lactic-co-glycolic acid) PLLA Poly(L-lactide) PLMA Poly(lauryl methacrylate) PMWS Post-weaning multi-systemic wasting syndrome PN Polymeric nanoparticles PPARγ Peroxisome proliferator-activated receptor gamma ppm Parts per million PPO Poly(p-phenylene oxide) PPRs Pattern recognition receptors PPTT Plasmonic photothermal therapy PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analyzes PROSPERO Prospective Register of Systematic Reviews PtBA Poly tert-butyl acrylate PTT Photothermal therapy PUBMED International database produced by the National Center for Biotechnology information PVP Polyvinylpyrrolidone PZQ Praziquantel QL Quantification limit qRT-PCR Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction R2 Correlation coefficient RAW 264.7 Mouse macrophage cell line RB Rose Bengal RECK Reversion-inducing-cysteine-rich protein with kazal motifs RIF Rifampicin RN Rio Grande do Norte State RNA Ribonucleic acid ROS Reactive oxygen species rpm Revolutions per minute RPMI 1640 Roswell Park Memorial Institute RS Rio Grande do Sul State RSD Relative standard deviation RT-PCR Reverse transcription polymerase chain reaction RVVC Recurrent vulvovaginal candidiasis S South latitude s Seconds SAOS-2 Human osteoblast-like cell line SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 SB Synthetic Broth SBA Santa Barbara Amorphous materials SBET Specific surface area 8 7 PBS Phosphate-buffered saline PCR Polymerase chain reaction PCV2 Porcine circovirus type 2 PDA Photodiode arrays PDI Polydispersity index PDMAEMA Poly(2-(dimethylamino)ethyl methacrylate) PDT Photodynamic therapy PEG Polyethylene glycol PEI Poly(ethylene imine) PEO Poly(ethylene oxide) PI3K Phosphoinositide 3-kinase PICO Population; intervention; comparation and outcome PK15 Pig kidney cell line PLGA Poly(lactic-co-glycolic acid) PLLA Poly(L-lactide) PLMA Poly(lauryl methacrylate) PMWS Post-weaning multi-systemic wasting syndrome PN Polymeric nanoparticles PPARγ Peroxisome proliferator-activated receptor gamma ppm Parts per million PPO Poly(p-phenylene oxide) PPRs Pattern recognition receptors PPTT Plasmonic photothermal therapy PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analyzes PROSPERO Prospective Register of Systematic Reviews PtBA Poly tert-butyl acrylate PTT Photothermal therapy PUBMED International database produced by the National Center for Biotechnology information PVP Polyvinylpyrrolidone PZQ Praziquantel QL Quantification limit qRT-PCR Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction R2 Correlation coefficient RAW 264.7 Mouse macrophage cell line RB Rose Bengal RECK Reversion-inducing-cysteine-rich protein with kazal motifs RIF Rifampicin RN Rio Grande do Norte State RNA Ribonucleic acid ROS Reactive oxygen species rpm Revolutions per minute RPMI 1640 Roswell Park Memorial Institute RS Rio Grande do Sul State RSD Relative standard deviation RT-PCR Reverse transcription polymerase chain reaction RVVC Recurrent vulvovaginal candidiasis S South latitude s Seconds SAOS-2 Human osteoblast-like cell line SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 SB Synthetic Broth SBA Santa Barbara Amorphous materials SBET Specific surface area 8 SBF Simulated body fluid SCFE Supercritical CO2 Extraction Scielo Scientific Eletronic Online Scopus International database produced by Elsevier SD Standard deviation SDA Sabouraud Dextrose Agar SDB Sabouraud Dextrose Broth SE Solvent extraction SEM Scanning electron microscopy SHI-1 Acute myelocytic leukemia cell line SIF Simulated intestinal fluid SLN Solid lipid nanoparticles SOD Superoxide dismutase SP São Paulo State SS Solid-state STAT Signal transducer and activator of transcription SV-80 Human lung fibroblast cell line SVF Simulated vaginal fluid SWAP Soluble adult worm antigen preparation TA Tannic acid TAC1 Zinc cluster transcription TBHP Tert-butyl hydroperoxide TEB Tebuconazole TEM Transmission electron microscopy TEOS Tetraethyl orthosilicate TG Thermogravimetric TG/DTA Thermogravimetry differential thermal analysis TGA Thermogravimetry analysis TH1 T helper 1 TMPES Trimethoxy-(2-phenylethyl)silane TMS Transmembrane spans TPP Tripolyphosphate pentasodium TSB Tryptic soy broth TTC Triphenyltetrazolium chloride TUD-1 Technical Delft University TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling TZM-bl Cell line derived from a HeLa cell clone U251MG Human glioblastoma cell line UAE Ultrasound-assisted extraction UBI29-41 Ubiquicidin UDCA Ursodeoxycholic acid UnTHCPSi NPs Carboxylic acid functionalized thermally hydrocarbonized porous silicon nanoparticles USP United States Pharmacopeia UV Ultraviolet UV-Vis Ultraviolet-Visible v Vacuum VAN Vancomycin VEEV Venezuelan equine encephalitis virus Vero cells Kidney epithelial cells extracted from an African green monkey Vk2/E6E7 Human vaginal epithelial cell line VSV Vesicular stomatitis virus VVC Vulvovaginal candidiasis W West longitude W Watts 8 9 9 W/O Water-in-oil w/w Weight in weight WHO World Health Organization Wnt Wingless-related integration site wt. Weight XTT 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]- 2H-tetrazolium-hydroxide Y-79 Human retinoblastoma cell linem ZnO@MSNs Zinc oxide nanoparticles into MSNs β Beta β-CD β-cyclodextrin μL Microliter 10 9 W/O Water-in-oil w/w Weight in weight WHO World Health Organization Wnt Wingless-related integration site wt. Weight XTT 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]- 2H-tetrazolium-hydroxide Y-79 Human retinoblastoma cell linem ZnO@MSNs Zinc oxide nanoparticles into MSNs β Beta β-CD β-cyclodextrin μL Microliter 11 CHAPTER 1 Introduction and aim of the thesis 10 11 12 1.Introduction As a result of the great global concern regarding the scarcity of research and political interventions to combat fungal infections and their growing problem about resistance to antifungal drugs currently used in therapy, the World Health Organization (WHO) recently made public a list of priority fungal pathogens. This list is divided into critical, high and medium priority, and it is observed that the genus Candida spp. is a threat to public health. Candida auris and Candida albicans were classified as critical priority, while Candida parapsilosis and Candida tropicalis were classified as medium priority (WHO, 2022). Candidiasis, the term used to refer to infections caused by this genus, can be superficial, cutaneous, mucosal or systemic (Mba & Nweze, 2020). Among the types of candidiasis, vulvovaginal candidiasis (VVC) must be highlighted, as is one of the most common causes of gynecological care nowadays, affecting at least once in life 75% of the world’s female population (Conte et al., 2023; Żyrek et al., 2021). Although this genus of fungus is commonly found in the vaginal mucosa microbiota of most healthy women, in a condition of commensalism, factors inherent to the host (e.g., microbiota changes and decreased production of endogenous estrogen), or even to the pathogen (like genetic and phenotypic variation), can break this balance, causing this fungus to become pathogenic (Becker & Sobel, 2023; Gaziano et al., 2023; Sun et al., 2023). When candida becomes pathogenic, it changes from the yeast to hyphae form, which invade the vaginal mucosa, triggering an immune response from the host mainly by inducing signaling cascades that lead to the production of pro-inflammatory cytokines and antimicrobial proteins by epithelial cells, macrophages and polymorphonuclear cells causing discomfort to the patient due to the symptoms of inflammation (Mahalingam et al., 2022). Among the main symptoms can be mentioned pruritus, edema, fissures and dysuria (Conte et al., 2023). Although different countries have their own protocols for the treatment of this disease, the common point between them is the use of the azoles class as one of the first therapeutic options of choice (British Association for Sexual Health and HIV, 2020; Farr et al., 2021; Hazra et al., 2022; Ministério da Saúde, 2022).Are some examples of drugs belonging to this class that are widely used in the treatment of VVC include: fluconazole, clotrimazole, miconazole, tioconazole, butoconazole and terconazole (Hazra et al., 2022). In addition to drugs derived from this class, such as benzydamine hydrochloride, an anti- inflammatory with antifungal properties as it is derived from indazole (Bossi et al., 2023). However, in the literature it is possible to observe several reports about Candida strains resistant to this class of drug, which appears as a challenge to be faced nowadays (Arrieta-Aguirre et al., 2023; Benhadj et al., 2024; Gerges et al., 2023). Therefore, despite seeking medical assistance, many women continue to have the infection despite have been treated, due to this issue of resistance, which can cause serious 12 Chapter 1 12 1.Introduction As a result of the great global concern regarding the scarcity of research and political interventions to combat fungal infections and their growing problem about resistance to antifungal drugs currently used in therapy, the World Health Organization (WHO) recently made public a list of priority fungal pathogens. This list is divided into critical, high and medium priority, and it is observed that the genus Candida spp. is a threat to public health. Candida auris and Candida albicans were classified as critical priority, while Candida parapsilosis and Candida tropicalis were classified as medium priority (WHO, 2022). Candidiasis, the term used to refer to infections caused by this genus, can be superficial, cutaneous, mucosal or systemic (Mba & Nweze, 2020). Among the types of candidiasis, vulvovaginal candidiasis (VVC) must be highlighted, as is one of the most common causes of gynecological care nowadays, affecting at least once in life 75% of the world’s female population (Conte et al., 2023; Żyrek et al., 2021). Although this genus of fungus is commonly found in the vaginal mucosa microbiota of most healthy women, in a condition of commensalism, factors inherent to the host (e.g., microbiota changes and decreased production of endogenous estrogen), or even to the pathogen (like genetic and phenotypic variation), can break this balance, causing this fungus to become pathogenic (Becker & Sobel, 2023; Gaziano et al., 2023; Sun et al., 2023). When candida becomes pathogenic, it changes from the yeast to hyphae form, which invade the vaginal mucosa, triggering an immune response from the host mainly by inducing signaling cascades that lead to the production of pro-inflammatory cytokines and antimicrobial proteins by epithelial cells, macrophages and polymorphonuclear cells causing discomfort to the patient due to the symptoms of inflammation (Mahalingam et al., 2022). Among the main symptoms can be mentioned pruritus, edema, fissures and dysuria (Conte et al., 2023). Although different countries have their own protocols for the treatment of this disease, the common point between them is the use of the azoles class as one of the first therapeutic options of choice (British Association for Sexual Health and HIV, 2020; Farr et al., 2021; Hazra et al., 2022; Ministério da Saúde, 2022).Are some examples of drugs belonging to this class that are widely used in the treatment of VVC include: fluconazole, clotrimazole, miconazole, tioconazole, butoconazole and terconazole (Hazra et al., 2022). In addition to drugs derived from this class, such as benzydamine hydrochloride, an anti- inflammatory with antifungal properties as it is derived from indazole (Bossi et al., 2023). However, in the literature it is possible to observe several reports about Candida strains resistant to this class of drug, which appears as a challenge to be faced nowadays (Arrieta-Aguirre et al., 2023; Benhadj et al., 2024; Gerges et al., 2023). Therefore, despite seeking medical assistance, many women continue to have the infection despite have been treated, due to this issue of resistance, which can cause serious 13 complications such as menstrual disorders, pelvic abscess, pelvic inflammatory disease, infertility, ectopic pregnancy and miscarriage (Gonçalves et al., 2016). In this context, the need to develop new therapeutic options is a growing trend and several strategies have been studied. Firstly, we can mention the development of new synthetic antifungals such as ibrexafungerp and oteseconazole. As oteseconazole belongs to the azoles class, is susceptible to the same mechanisms of fungal resistance as its class, however, once it is a new therapeutic option, there are still no clinical reports about it being therefore an option for the treatment of this disease (Sobel, 2023a). Another interesting alternative is the use of innovative pharmaceutical forms, such as chitosan- based sponges (biodegradable/dissolvable) containing clotrimazole and sprayable thermosensitive benzydamine hydrogel (an anti-inflammatory with known antifungal activity) (Arpa et al., 2023; Martins et al., 2023). The association of drugs is already well established in the therapy (azoles and other classes of antifungal) with nanotechnology in order to increase their therapeutic activity, bypassing the resistance issue is also an alternative that has been widely used like itraconazole-based nanosuspension, clotrimazole-loaded cationic N-(2-hydroxy)-propyl-3-trimethylammonium, O-palmitoyl chitosan nanoparticles and amphotericin B-loaded nanoemulsion (Facchinatto et al., 2021; Marena et al., 2022; Parsana et al., 2023). Although there are other options nowadays, the last strategy that needs to be highlighted is the use of products of natural origin, like lycopene and curcumin, (alone or in association) as they generally have effectiveness, considerable biocompatibility and low toxicity; however, many of them have limitations (e.g., low aqueous solubility and low absorption) that limit their use in therapy. For this reason, nanotechnology is often also a protagonist when talking about natural products for biological applications (Marena et al., 2022). As example, can be cited fluconazole and propolis co-encapsulated in chitosan nanoparticles; hypericin-loaded nanostructured lipid carriers and chitosan-based system with Scutellariae baicalensis radix extract (Chanaj-Kaczmarek et al., 2022; da Silva et al., 2023; Sato et al., 2023). As a result of this scenario, this thesis sought to develop an ideal formulation based on natural products and nanotechnology in order to combat this growing issue of resistance. However, it is important to highlight that the process of developing a formulation goes through several stages, from the process of conceiving the study hypothesis to the obtainment of a final product, which may or may not prove promising for the purpose studied. To reach the top of the trajectory, that is, to obtain a promising formulation to treat VVC, several steps must be completed. In this case, to develop a nano- in-nano system dual-loaded with curcumin and benzydamine hydrochloride dispersed in a thermoresponsive hydrogel, it was necessary to go through several prior steps, such as the production 12 13 1 Introduction and aim of the thesis 14 of review articles to learn more about the topics and the production of a preliminary nanotechnological system, because that’s what science is about: attempts based on scientific knowledge. 2. Summary of the thesis content According to the Dutch Institute for Healthcare Research, in 2022 urogenital candidiasis in women showed a prevalence of 19.2 per 1000 inhabitants (Dutch Institute for Research in Health Care, 2022). In Brazil, both official data and those published in the literature are obsolete and incomplete, which makes it difficult to understand the epidemiological profile of this disease throughout the Brazilian territory, information that is essential for establishing public policy plans for disease prevention and combat. Due to this shortcoming, a systematic review with meta-analysis (registered in the International Prospective Register of Systematic Reviews: PROSPERO 2020 CRD42020181695) was carried out in Chapter 2. Although Candida albicans is the specie most frequently isolated from vulvovaginal candidiasis samples, non-albicans species can also be isolated, with C. glabrata and C. tropicalis being the most prevalent ones (Chen et al., 2023; Seck et al., 2023). Although not prevalent, emphasis needs to be placed on cases of VVC caused by C. auris, since this new emerging yeast is already multidrug- resistant and difficult to eradicate (Krishnasamy et al., 2021; Kumar et al., 2015; Mesini et al., 2021). Despite being considered a great challenge, little is still known about this specie in the scientific literature, which is why Chapter 3 addresses the state of the art of what is known so far about this new specie. Turning the attention to the most prevalent one, C. albicans is a fungus that inhabits the vulvovaginal mucosa as a commensal microorganism, in the form of yeast; however, as it is a dimorphic fungus, it can change to its pathogenic state in host situations, such as elevation of vaginal pH, microbiota disruption (due the use of antibiotics for example), use of high-estrogen oral contraceptives and hormone replacement, diabetes mellitus and impairment of the immune system (Czechowicz et al., 2022; Peters et al., 2014). This transition to the pathogenic form begins with the change from yeast to hyphae (which does not occur in other species such as C. glabrata for example), which invade and damage the host tissue. The fungal components are then recognized by the presenting cells of innate antigens, which triggers the activation of nuclear factor kappa B (NF-kB) resulting in the release of pro-inflammatory cytokines and reactive oxygen species (ROS), that is, the pathogenicity of C. albicans leads to an inflammatory response in the host organism (Mahalingam et al., 2022; Peters et al., 2014). 14 Chapter 1 14 of review articles to learn more about the topics and the production of a preliminary nanotechnological system, because that’s what science is about: attempts based on scientific knowledge. 2. Summary of the thesis content According to the Dutch Institute for Healthcare Research, in 2022 urogenital candidiasis in women showed a prevalence of 19.2 per 1000 inhabitants (Dutch Institute for Research in Health Care, 2022). In Brazil, both official data and those published in the literature are obsolete and incomplete, which makes it difficult to understand the epidemiological profile of this disease throughout the Brazilian territory, information that is essential for establishing public policy plans for disease prevention and combat. Due to this shortcoming, a systematic review with meta-analysis (registered in the International Prospective Register of Systematic Reviews: PROSPERO 2020 CRD42020181695) was carried out in Chapter 2. Although Candida albicans is the specie most frequently isolated from vulvovaginal candidiasis samples, non-albicans species can also be isolated, with C. glabrata and C. tropicalis being the most prevalent ones (Chen et al., 2023; Seck et al., 2023). Although not prevalent, emphasis needs to be placed on cases of VVC caused by C. auris, since this new emerging yeast is already multidrug- resistant and difficult to eradicate (Krishnasamy et al., 2021; Kumar et al., 2015; Mesini et al., 2021). Despite being considered a great challenge, little is still known about this specie in the scientific literature, which is why Chapter 3 addresses the state of the art of what is known so far about this new specie. Turning the attention to the most prevalent one, C. albicans is a fungus that inhabits the vulvovaginal mucosa as a commensal microorganism, in the form of yeast; however, as it is a dimorphic fungus, it can change to its pathogenic state in host situations, such as elevation of vaginal pH, microbiota disruption (due the use of antibiotics for example), use of high-estrogen oral contraceptives and hormone replacement, diabetes mellitus and impairment of the immune system (Czechowicz et al., 2022; Peters et al., 2014). This transition to the pathogenic form begins with the change from yeast to hyphae (which does not occur in other species such as C. glabrata for example), which invade and damage the host tissue. The fungal components are then recognized by the presenting cells of innate antigens, which triggers the activation of nuclear factor kappa B (NF-kB) resulting in the release of pro-inflammatory cytokines and reactive oxygen species (ROS), that is, the pathogenicity of C. albicans leads to an inflammatory response in the host organism (Mahalingam et al., 2022; Peters et al., 2014). 15 In view of the emergence of C. albicans strains resistant to currently available therapies, mainly azoles, and that the inflammation generated by the infiltration of fungal hyphae in the vulvovaginal mucosa leads to the appearance of symptoms, so painful and uncomfortable for the patient, the study of new therapeutic options is increasingly necessary, especially combining antifungal and anti- inflammatory actions (Sobel, 2023b; Sobel et al., 2022). In this context, drugs of natural origin have been explored as promising options to overcome the problem of fungal resistance (Marena et al., 2022). Among the various natural molecules explored today, lycopene stands out, a carotenoid extracted mainly from tomatoes that has several therapeutic activities, such as antifungal and anti-inflammatory, being therefore desirable for the treatment of VVC (Amorim et al., 2022). However, in addition to low aqueous solubility, this compound has other characteristics that limit its application in therapy, such as sensitivity to light, temperature and oxygen, factors that can lead to isomerism of the molecule, which is not desirable since the different lycopene isomers have different bioavailability and bioaccessibility (Falsafi et al., 2022; Li et al., 2023). This highlights the importance of knowing its physicochemical characteristics and preserving it from extraction until the moment of its application to ensure the maintenance of its biological properties. Thus, Chapter 4 highlights the entire route that lycopene takes from its cultivation and extraction to its use as a nutraceutical and possible association with nanotechnology. Lycopene association with nanotechnology, like nanoparticles, is an interesting strategy to not only preserve its physicochemical characteristics but also increase its aqueous solubility. The small size of nanoparticles associated with their large surface area-to-volume make them an interesting strategy for drug delivery. It is also worth highlighting that this association also provides increased biological efficacy, reduced dosage and fewer adverse effects of the active molecule (Falsafi et al., 2022; Manocha et al., 2022). According to their composition, nanoparticles are generally categorized into four classes, inorganic, organic, carbon-based and hybrid (Joudeh & Linke, 2022). For the process of developing a nanoformulation, it is essential to study and define the desirable characteristics for the appropriate choice of the nanocarrier. Hence, Chapter 5 reinforces the lack of work exploring the association of lycopene with inorganic nanoparticles, highlighting a new topic for study. This chapter also highlights what is in the literature regarding analytical methods for detecting this drug, a key topic for the development of a formulation as it makes it possible to know the amount of drug incorporated and released by the system, thus allowing dose adjustment. Despite the various inorganic materials used as nanocarriers, it is worth highlighting the silica-based ones, more specifically mesoporous silica nanoparticles (MSN), suitable for biological applications as they have low immunogenicity and good biocompatibility. Furthermore, its large surface area and pore volume give to this nanocarrier not only a high carrying capacity for small or large molecules, 14 15 1 Introduction and aim of the thesis 16 contributing positively to its behavior in the biological environment, but also increase its cellular absorption (Miguel Sábio et al., 2023; Parra‐Nieto et al., 2021; Yang et al., 2020). To highlight whether this type of nanoparticle could be an intelligent strategy to combat infectious diseases, such as VVC, specially caused by multidrug resistant microorganisms, Chapter 6 seeks to explore this topic further. The abovementioned desirable characteristics, associated with the growing trend of using this type of nanoparticle to combat infectious diseases, made it the choice to be associated with lycopene. The development and characterization process of lycopene-loaded MSN are described in Chapters 7 and 8. An important detail that needs to be considered during the MSN synthesis is the availability of the mesopores for drug loading. It is possible to observe studies in the literature where physicochemical characterization analyzes point to residues of organic matter (e.g., cetyltrimethylammonium bromide, CTAB) even after MSNs have undergone removal processes, which can compromise the drug incorporation process (Javdani et al., 2020; Joyce et al., 2020; Pongchaikul et al., 2023; Prem et al., 2023; Wu et al., 2021). In this regard, Chapter 9 provides an extensive and unprecedented discussion about the use of this surfactant in the synthesis of MSN and its toxicity in eukaryotic and prokaryotic cells, since CTAB is a quaternary ammonium compound (Li et al., 2023). Additionally, the issue of the CTAB residues in MSN led to the execution of the toxicity assessment in Galleria mellonella, addressed in Chapter 8. Chapter 8 also addresses the antifungal activity of loaded and free lycopene (see Appendix 1 for lycopene anti-inflammatory activity). In addition to lycopene, another product of natural origin that has increasingly receiving considerable attention due to its biological activities, like anti-inflammatory and antifungal, is curcumin (Gayathri et al., 2023; Obeid et al., 2023). Diferuloylmethane, or curcumin as it is more widely known, is a yellow-colored polyphenol found primarily in the rhizome of Curcuma longa (L) and other Curcuma spp (Sharifi-Rad et al., 2020). Like lycopene, this compound also has limitations that hinder its application in therapy, such as limited water solubility, poor absorption and low bioavailability, rapid metabolism and sensitivity to light (Obeid et al., 2023). Chapter 10 highlights the importance to know and preserve curcumin physicochemical characteristics from its cultivation and extraction towards its biological application and association with nanotechnology, crucial to overcome the cited limitations. The association of this molecule, with consolidated antifungal activity, with an anti-inflammatory already consolidated in therapy, especially when dealing with gynecological inflammations, such as benzydamine hydrochloride, in a nanosystem that allows the simultaneous incorporation of both drugs proves to be an interesting strategy (Araujo et al., 2020; Di Stefano et al., 2020; Hussain et al., 2022). From this perspective, Chapter 11 describes the development of a hybrid nanoparticle (nano-in-nano) by microfluidics composed of MSN and a lipid-coating. Lipids were chosen for the manufacture of 16 Chapter 1 16 contributing positively to its behavior in the biological environment, but also increase its cellular absorption (Miguel Sábio et al., 2023; Parra‐Nieto et al., 2021; Yang et al., 2020). To highlight whether this type of nanoparticle could be an intelligent strategy to combat infectious diseases, such as VVC, specially caused by multidrug resistant microorganisms, Chapter 6 seeks to explore this topic further. The abovementioned desirable characteristics, associated with the growing trend of using this type of nanoparticle to combat infectious diseases, made it the choice to be associated with lycopene. The development and characterization process of lycopene-loaded MSN are described in Chapters 7 and 8. An important detail that needs to be considered during the MSN synthesis is the availability of the mesopores for drug loading. It is possible to observe studies in the literature where physicochemical characterization analyzes point to residues of organic matter (e.g., cetyltrimethylammonium bromide, CTAB) even after MSNs have undergone removal processes, which can compromise the drug incorporation process (Javdani et al., 2020; Joyce et al., 2020; Pongchaikul et al., 2023; Prem et al., 2023; Wu et al., 2021). In this regard, Chapter 9 provides an extensive and unprecedented discussion about the use of this surfactant in the synthesis of MSN and its toxicity in eukaryotic and prokaryotic cells, since CTAB is a quaternary ammonium compound (Li et al., 2023). Additionally, the issue of the CTAB residues in MSN led to the execution of the toxicity assessment in Galleria mellonella, addressed in Chapter 8. Chapter 8 also addresses the antifungal activity of loaded and free lycopene (see Appendix 1 for lycopene anti-inflammatory activity). In addition to lycopene, another product of natural origin that has increasingly receiving considerable attention due to its biological activities, like anti-inflammatory and antifungal, is curcumin (Gayathri et al., 2023; Obeid et al., 2023). Diferuloylmethane, or curcumin as it is more widely known, is a yellow-colored polyphenol found primarily in the rhizome of Curcuma longa (L) and other Curcuma spp (Sharifi-Rad et al., 2020). Like lycopene, this compound also has limitations that hinder its application in therapy, such as limited water solubility, poor absorption and low bioavailability, rapid metabolism and sensitivity to light (Obeid et al., 2023). Chapter 10 highlights the importance to know and preserve curcumin physicochemical characteristics from its cultivation and extraction towards its biological application and association with nanotechnology, crucial to overcome the cited limitations. The association of this molecule, with consolidated antifungal activity, with an anti-inflammatory already consolidated in therapy, especially when dealing with gynecological inflammations, such as benzydamine hydrochloride, in a nanosystem that allows the simultaneous incorporation of both drugs proves to be an interesting strategy (Araujo et al., 2020; Di Stefano et al., 2020; Hussain et al., 2022). From this perspective, Chapter 11 describes the development of a hybrid nanoparticle (nano-in-nano) by microfluidics composed of MSN and a lipid-coating. Lipids were chosen for the manufacture of 17 this hybrid nanoparticle because they are highly biocompatible, biodegradable, has low immunogenicity and are known to increase the aqueous solubility of hydrophobic drugs. In addition to being clinically approved by the European Medicines Agency (EMA) and Food and Drug Administration (FDA) for therapeutic applications (Giordani et al., 2023; Nsairat et al., 2022). To synthesize this particle, microfluidics was the technique of choice because it is highly controllable, reproducible (maintaining homogeneity), can improve the drug encapsulation efficiency and is scalable (Fontana et al., 2019; Liu et al., 2020; Martins & Santos, 2023). It is worth highlighting that in this chapter, a thermoresponsive hydrogel was also used as an intelligent strategy to enable the topical application of the hybrid nanoparticle in vivo, since it is liquid at room temperature, making it easy to apply, and gel at body temperature which associated with the mucoadhesive character of chitosan enables the maintenance of the formulation in the target site (Araujo et al., 2021; dos Santos et al., 2020; Kolawole & Cook, 2023). Due to its thermoresponsive characteristic, poloxamer has been increasingly used for topical formulations where this behavior is desirable, such as the vaginal environment (Su et al., 2023; Sukmawaty et al., 2023). Finally, Chapter 12 provides an overview of the general aspects and biological behavior of this polymer as well as its application for controlled drug release in the vaginal environment. 3. Aim of this thesis Given the high prevalence of VVC and the growing problem of resistance to antifungals commonly used in the clinical practice, it is urgent to develop new therapeutic options to not only combat its causative agent but also to bring better living conditions to the affected patients. In this regard, the aim of this thesis was to a develop drug delivery nanoformulation, associating natural product with nanotechnology to treat, VVC caused by resistant fungi strains. To achieve this, an in- depth study of the literature was necessary to, based on scientific knowledge, choose, and associate the best strategies in order to produce an innovative formulation. As the first hypothesis is not always ideal one to reach the ideal innovative formulation, two nanosystems with different strategies but with some points in common were developed and evaluated in vitro and in vivo. 16 17 1 Introduction and aim of the thesis 18 4. References Amorim, A. das G. N., Vasconcelos, A. G., Souza, J., Oliveira, A., Gullón, B., de Souza de Almeida Leite, J. R., & Pintado, M. (2022). Bio-Availability, Anticancer Potential, and Chemical Data of Lycopene: An Overview and Technological Prospecting. Antioxidants, 11(2), Article 360. https://doi.org/10.3390/antiox11020360 Araujo, V. H. S., de Souza, M. P. C., Carvalho, G. C., Duarte, J. L., & Chorilli, M. (2021). Chitosan-based systems aimed at local applications for vaginal infections. Carbohydrate Polymers, 261, Article 117919. https://doi.org/10.1016/j.carbpol.2021.117919 Araujo, V. H. S., Duarte, J. L., Carvalho, G. C., Silvestre, A. L. P., Fonseca-Santos, B., Marena, G. D., Ribeiro, T. de C., dos Santos Ramos, M. A., Bauab, T. M., & Chorilli, M. (2020). Nanosystems against candidiasis: a review of studies performed over the last two decades. Critical Reviews in Microbiology, 46(5), 508-547. https://doi.org/10.1080/1040841X.2020.1803208 Arpa, M. D., Kesmen, E. E., & Biltekin, S. N. (2023). Novel Sprayable Thermosensitive Benzydamine Hydrogels for Topical Application: Development, Characterization, and In Vitro Biological Activities. AAPS PharmSciTech, 24(8), Article 214. https://doi.org/10.1208/s12249-023-02674-w Arrieta-Aguirre, I., Menéndez-Manjón, P., Carrano, G., Diez, A., Fernandez-de-Larrinoa, Í., & Moragues, M.-D. (2023). Molecular Identification of Fungal Species through Multiplex-qPCR to Determine Candidal Vulvovaginitis and Antifungal Susceptibility. Journal of Fungi, 9(12), Article 1145. https://doi.org/10.3390/jof9121145 Becker, M., & Sobel, R. (2023). Vulvovaginal Candidiasis in Postmenopausal Women. Current Infectious Disease Reports, 25(4), 61–66. https://doi.org/10.1007/s11908-023-00801-z Benhadj, M., Menasria, T., & Ranque, S. (2024). MALDI-TOF mass spectrometry identification and antifungal susceptibility testing of yeasts causing vulvovaginal candidiasis (VVC) in Tebessa (Northeastern Algeria). Annales de Biologie Clinique, 81(6), 576-584. https://doi.org/10.1684/abc.2023.1852 Bossi, P., Gurizzan, C., Guntinas-Lichius, O., Hainarosie, R., & Lancini, D. (2023). The present and the future of benzydamine: expert opinion paper. Frontiers in Oral Health, 4, Article 1191319. https://doi.org/10.3389/froh.2023.1191319 British Association for Sexual Health and HIV. (2020). British Association for Sexual Health and HIV national guideline for the management of vulvovaginal candidiasis (2019). International Journal of STD & AIDS, 31(12), 1124–1144. https://doi.org/10.1177/0956462420943034 Chanaj-Kaczmarek, J., Rosiak, N., Szymanowska, D., Rajewski, M., Wender-Ozegowska, E., & Cielecka-Piontek, J. (2022). The Chitosan-Based System with Scutellariae baicalensis radix Extract for the Local Treatment of Vaginal Infections. Pharmaceutics, 14(4), Article 740. https://doi.org/10.3390/pharmaceutics14040740 Chen, Z., Jin, J., Chen, H., Chen, Y., & Feng, S. (2023). The bacterial communities in vagina of different Candida species-associated vulvovaginal candidiasis. Microbial Pathogenesis, 177, Article 106037. https://doi.org/10.1016/j.micpath.2023.106037 Conte, J., Parize, A. L., & Caon, T. (2023). Advanced Solid Formulations For Vulvovaginal Candidiasis. Pharmaceutical Research, 40(2), 593–610. https://doi.org/10.1007/s11095-022-03441-5 Czechowicz, P., Nowicka, J., & Gościniak, G. (2022). Virulence Factors of Candida spp. and Host Immune Response Important in the Pathogenesis of Vulvovaginal Candidiasis. International Journal of Molecular Sciences, 23(11), Article 5895. https://doi.org/10.3390/ijms23115895 da Silva, J. T., Dantas de Sousa, P. H., Costa, A. F., de Menezes, L. B., Alves, S. F., Pellegrini, F., & Amaral, A. C. (2023). Fluconazole and propolis co- encapsulated in chitosan nanoparticles for the treatment of vulvovaginal candidiasis in a murine model. Medical Mycology, 61(11), Article myad113. https://doi.org/10.1093/mmy/myad113 Di Stefano, A. F. D., Radicioni, M. M., Vaccani, A., Caccia, G., Focanti, F., Salvatori, E., Pelacchi, F., Picollo, R., Rosignoli, M. T., Olivieri, S., & others. (2020). Phase I Study in Healthy Women of a Novel Antimycotic Vaginal Ovule Combining Econazole and Benzydamine. Infectious Diseases in Obstetrics and Gynecology, 2020, 1–11. https://doi.org/10.1155/2020/7201840 dos Santos, A. M., Carvalho, S. G., Araujo, V. H. S., Carvalho, G. C., Gremião, M. P. D., & Chorilli, M. (2020). Recent advances in hydrogels as strategy for drug delivery intended to vaginal infections. International Journal of Pharmaceutics, 590, Article 119867. https://doi.org/10.1016/j.ijpharm.2020.119867 Dutch Institute for Research in Health Care. (2022). Jaarcijfers aandoeningen - Huisartsenregistraties. Nivel. https://www.nivel.nl/nl/nivel- zorgregistraties-eerste-lijn/cijfers-over-aandoeningen/jaarcijfers-aandoeningen-huisartsenregistraties Facchinatto, W. M., Galante, J., Mesquita, L., Silva, D. S., Martins dos Santos, D., Moraes, T. B., Campana-Filho, S. P., Colnago, L. A., Sarmento, B., & das Neves, J. (2021). Clotrimazole-loaded N-(2-hydroxy)-propyl-3-trimethylammonium, O-palmitoyl chitosan nanoparticles for topical treatment of vulvovaginal candidiasis. Acta Biomaterialia, 125, 312–321. https://doi.org/10.1016/j.actbio.2021.02.029 Falsafi, S. R., Rostamabadi, H., Babazadeh, A., Tarhan, Ö., Rashidinejad, A., Boostani, S., Khoshnoudi-Nia, S., Akbari-Alavijeh, S., Shaddel, R., & Jafari, S. M. (2022). Lycopene nanodelivery systems; recent advances. Trends in Food Science & Technology, 119, 378–399. https://doi.org/10.1016/j.tifs.2021.12.016 Farr, A., Effendy, I., Frey Tirri, B., Hof, H., Mayser, P., Petricevic, L., Ruhnke, M., Schaller, M., Schaefer, A. P. A., Sustr, V., & others. (2021). Guideline: Vulvovaginal candidosis (AWMF 015/072, level S2k). Mycoses, 64(6), 583–602. https://doi.org/10.1111/myc.13248 Fontana, F., Martins, J. P., Torrieri, G., & Santos, H. A. (2019). Nuts and Bolts: Microfluidics for the Production of Biomaterials. Advanced Materials Technologies, 4(6), Article 1800611. https://doi.org/10.1002/admt.201800611 Gayathri, K., Bhaskaran, M., Selvam, C., & Thilagavathi, R. (2023). Nano formulation approaches for curcumin delivery- a review. Journal of Drug Delivery Science and Technology, 82, Article 104326. https://doi.org/10.1016/j.jddst.2023.104326 18 Chapter 1 18 4. References Amorim, A. das G. N., Vasconcelos, A. G., Souza, J., Oliveira, A., Gullón, B., de Souza de Almeida Leite, J. R., & Pintado, M. (2022). Bio-Availability, Anticancer Potential, and Chemical Data of Lycopene: An Overview and Technological Prospecting. Antioxidants, 11(2), Article 360. https://doi.org/10.3390/antiox11020360 Araujo, V. H. S., de Souza, M. P. C., Carvalho, G. C., Duarte, J. L., & Chorilli, M. (2021). Chitosan-based systems aimed at local applications for vaginal infections. Carbohydrate Polymers, 261, Article 117919. https://doi.org/10.1016/j.carbpol.2021.117919 Araujo, V. H. S., Duarte, J. L., Carvalho, G. C., Silvestre, A. L. P., Fonseca-Santos, B., Marena, G. D., Ribeiro, T. de C., dos Santos Ramos, M. A., Bauab, T. M., & Chorilli, M. (2020). Nanosystems against candidiasis: a review of studies performed over the last two decades. Critical Reviews in Microbiology, 46(5), 508-547. https://doi.org/10.1080/1040841X.2020.1803208 Arpa, M. D., Kesmen, E. E., & Biltekin, S. N. (2023). Novel Sprayable Thermosensitive Benzydamine Hydrogels for Topical Application: Development, Characterization, and In Vitro Biological Activities. AAPS PharmSciTech, 24(8), Article 214. https://doi.org/10.1208/s12249-023-02674-w Arrieta-Aguirre, I., Menéndez-Manjón, P., Carrano, G., Diez, A., Fernandez-de-Larrinoa, Í., & Moragues, M.-D. (2023). Molecular Identification of Fungal Species through Multiplex-qPCR to Determine Candidal Vulvovaginitis and Antifungal Susceptibility. Journal of Fungi, 9(12), Article 1145. https://doi.org/10.3390/jof9121145 Becker, M., & Sobel, R. (2023). Vulvovaginal Candidiasis in Postmenopausal Women. Current Infectious Disease Reports, 25(4), 61–66. https://doi.org/10.1007/s11908-023-00801-z Benhadj, M., Menasria, T., & Ranque, S. (2024). MALDI-TOF mass spectrometry identification and antifungal susceptibility testing of yeasts causing vulvovaginal candidiasis (VVC) in Tebessa (Northeastern Algeria). Annales de Biologie Clinique, 81(6), 576-584. https://doi.org/10.1684/abc.2023.1852 Bossi, P., Gurizzan, C., Guntinas-Lichius, O., Hainarosie, R., & Lancini, D. (2023). The present and the future of benzydamine: expert opinion paper. Frontiers in Oral Health, 4, Article 1191319. https://doi.org/10.3389/froh.2023.1191319 British Association for Sexual Health and HIV. (2020). British Association for Sexual Health and HIV national guideline for the management of vulvovaginal candidiasis (2019). International Journal of STD & AIDS, 31(12), 1124–1144. https://doi.org/10.1177/0956462420943034 Chanaj-Kaczmarek, J., Rosiak, N., Szymanowska, D., Rajewski, M., Wender-Ozegowska, E., & Cielecka-Piontek, J. (2022). The Chitosan-Based System with Scutellariae baicalensis radix Extract for the Local Treatment of Vaginal Infections. Pharmaceutics, 14(4), Article 740. https://doi.org/10.3390/pharmaceutics14040740 Chen, Z., Jin, J., Chen, H., Chen, Y., & Feng, S. (2023). The bacterial communities in vagina of different Candida species-associated vulvovaginal candidiasis. Microbial Pathogenesis, 177, Article 106037. https://doi.org/10.1016/j.micpath.2023.106037 Conte, J., Parize, A. L., & Caon, T. (2023). Advanced Solid Formulations For Vulvovaginal Candidiasis. Pharmaceutical Research, 40(2), 593–610. https://doi.org/10.1007/s11095-022-03441-5 Czechowicz, P., Nowicka, J., & Gościniak, G. (2022). Virulence Factors of Candida spp. and Host Immune Response Important in the Pathogenesis of Vulvovaginal Candidiasis. International Journal of Molecular Sciences, 23(11), Article 5895. https://doi.org/10.3390/ijms23115895 da Silva, J. T., Dantas de Sousa, P. H., Costa, A. F., de Menezes, L. B., Alves, S. F., Pellegrini, F., & Amaral, A. C. (2023). Fluconazole and propolis co- encapsulated in chitosan nanoparticles for the treatment of vulvovaginal candidiasis in a murine model. Medical Mycology, 61(11), Article myad113. https://doi.org/10.1093/mmy/myad113 Di Stefano, A. F. D., Radicioni, M. M., Vaccani, A., Caccia, G., Focanti, F., Salvatori, E., Pelacchi, F., Picollo, R., Rosignoli, M. T., Olivieri, S., & others. (2020). Phase I Study in Healthy Women of a Novel Antimycotic Vaginal Ovule Combining Econazole and Benzydamine. Infectious Diseases in Obstetrics and Gynecology, 2020, 1–11. https://doi.org/10.1155/2020/7201840 dos Santos, A. M., Carvalho, S. G., Araujo, V. H. S., Carvalho, G. C., Gremião, M. P. D., & Chorilli, M. (2020). Recent advances in hydrogels as strategy for drug delivery intended to vaginal infections. International Journal of Pharmaceutics, 590, Article 119867. https://doi.org/10.1016/j.ijpharm.2020.119867 Dutch Institute for Research in Health Care. (2022). Jaarcijfers aandoeningen - Huisartsenregistraties. Nivel. https://www.nivel.nl/nl/nivel- zorgregistraties-eerste-lijn/cijfers-over-aandoeningen/jaarcijfers-aandoeningen-huisartsenregistraties Facchinatto, W. M., Galante, J., Mesquita, L., Silva, D. S., Martins dos Santos, D., Moraes, T. B., Campana-Filho, S. P., Colnago, L. A., Sarmento, B., & das Neves, J. (2021). Clotrimazole-loaded N-(2-hydroxy)-propyl-3-trimethylammonium, O-palmitoyl chitosan nanoparticles for topical treatment of vulvovaginal candidiasis. Acta Biomaterialia, 125, 312–321. https://doi.org/10.1016/j.actbio.2021.02.029 Falsafi, S. R., Rostamabadi, H., Babazadeh, A., Tarhan, Ö., Rashidinejad, A., Boostani, S., Khoshnoudi-Nia, S., Akbari-Alavijeh, S., Shaddel, R., & Jafari, S. M. (2022). Lycopene nanodelivery systems; recent advances. Trends in Food Science & Technology, 119, 378–399. https://doi.org/10.1016/j.tifs.2021.12.016 Farr, A., Effendy, I., Frey Tirri, B., Hof, H., Mayser, P., Petricevic, L., Ruhnke, M., Schaller, M., Schaefer, A. P. A., Sustr, V., & others. (2021). Guideline: Vulvovaginal candidosis (AWMF 015/072, level S2k). Mycoses, 64(6), 583–602. https://doi.org/10.1111/myc.13248 Fontana, F., Martins, J. P., Torrieri, G., & Santos, H. A. (2019). Nuts and Bolts: Microfluidics for the Production of Biomaterials. Advanced Materials Technologies, 4(6), Article 1800611. https://doi.org/10.1002/admt.201800611 Gayathri, K., Bhaskaran, M., Selvam, C., & Thilagavathi, R. (2023). Nano formulation approaches for curcumin delivery- a review. Journal of Drug Delivery Science and Technology, 82, Article 104326. https://doi.org/10.1016/j.jddst.2023.104326 19 Gaziano, R., Sabbatini, S., & Monari, C. (2023). The Interplay between Candida albicans, Vaginal Mucosa, Host Immunity and Resident Microbiota in Health and Disease: An Overview and Future Perspectives. Microorganisms, 11(5), Article 1211. https://doi.org/10.3390/microorganisms11051211 Gerges, M. A., Fahmy, Y. A., Hosny, T., Gandor, N. H., Mohammed, S. Y., Mohamed, T. M. A., Abdelmoteleb, N. E. M., & Esmaeel, N. E. (2023). Biofilm Formation and Aspartyl Proteinase Activity and Their Association with Azole Resistance Among Candida albicans Causing Vulvovaginal Candidiasis, Egypt. Infection and Drug Resistance, 16, 5283–5293. https://doi.org/10.2147/IDR.S420580 Giordani, S., Marassi, V., Zattoni, A., Roda, B., & Reschiglian, P. (2023). Liposomes characterization for market approval as pharmaceutical products: Analytical methods, guidelines and standardized protocols. Journal of Pharmaceutical and Biomedical Analysis, 236, Article 115751. https://doi.org/10.1016/j.jpba.2023.115751 Gonçalves, B., Ferreira, C., Alves, C. T., Henriques, M., Azeredo, J., & Silva, S. (2016). Vulvovaginal candidiasis: Epidemiology, microbiology and risk factors. Critical Reviews in Microbiology, 42(6), 905–927. https://doi.org/10.3109/1040841X.2015.1091805 Hazra, A., Collison, M. W., & Davis, A. M. (2022). CDC Sexually Transmitted Infections Treatment Guidelines, 2021. Jama 327(9), 870-871. https://doi.org/10.1001/jama.2022.1246 Hussain, Y., Alam, W., Ullah, H., Dacrema, M., Daglia, M., Khan, H., & Arciola, C. R. (2022). Antimicrobial Potential of Curcumin: Therapeutic Potential and Challenges to Clinical Applications. Antibiotics, 11(3), Article 322. https://doi.org/10.3390/antibiotics11030322 Javdani, H., Khosravi, R., Etemad, L., Moshiri, M., Zarban, A., & Hanafi-Bojd, M. Y. (2020). Tannic acid-templated mesoporous silica nanoparticles as an effective treatment in acute ferrous sulfate poisoning. Microporous and Mesoporous Materials, 307, Article 110486. https://doi.org/10.1016/j.micromeso.2020.110486 Joudeh, N., & Linke, D. (2022). Nanoparticle classification, physicochemical properties, characterization, and applications: a comprehensive review for biologists. Journal of Nanobiotechnology, 20(1), Article 262. https://doi.org/10.1186/s12951-022-01477-8 Joyce, P., Ulmefors, H., Maghrebi, S., Subramaniam, S., Wignall, A., Jõemetsa, S., Höök, F., & Prestidge, C. A. (2020). Enhancing the Cellular Uptake and Antibacterial Activity of Rifampicin through Encapsulation in Mesoporous Silica Nanoparticles. Nanomaterials, 10(4), Article 815. https://doi.org/10.3390/nano10040815 Kolawole, O. M., & Cook, M. T. (2023). In situ gelling drug delivery systems for topical drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 184, 36–49. https://doi.org/10.1016/j.ejpb.2023.01.007 Krishnasamy, L., Senthilganesh, J., Saikumar, C., & Nithyanand, P. (2021). Biofilm-forming fluconazole-resistant Candida auris causing vulvovaginal candidiasis in an immunocompetent patient: A case report. Asian Pacific Journal of Tropical Medicine, 14(2), 94-96. https://doi.org/10.4103/1995-7645.306768 Kumar, D., Banerjee, T., Pratap, C. B., & Tilak, R. (2015). Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis. The Journal of Infection in Developing Countries, 9(4), 435–437. https://doi.org/10.3855/jidc.4582 Li, Y., Cui, Z., & Hu, L. (2023). Recent technological strategies for enhancing the stability of lycopene in processing and production. Food Chemistry, 405, Article 134799. https://doi.org/10.1016/j.foodchem.2022.134799 Li, Y., Li, B., Guo, X., Wang, H., & Cheng, L. (2023). Applications of quaternary ammonium compounds in the prevention and treatment of oral diseases: State-of-the-art and future directions. Journal of Dentistry, 137, Article 104678. https://doi.org/10.1016/j.jdent.2023.104678 Liu, Z., Fontana, F., Python, A., Hirvonen, J. T., & Santos, H. A. (2020). Microfluidics for Production of Particles: Mechanism, Methodology, and Applications. Small, 16(9), Article 1904673. https://doi.org/10.1002/smll.201904673 Mahalingam, S. S., Jayaraman, S., & Pandiyan, P. (2022). Fungal Colonization and Infections—Interactions with Other Human Diseases. Pathogens, 11(2), Article 212. https://doi.org/10.3390/pathogens11020212 Manocha, S., Dhiman, S., Grewal, A. S., & Guarve, K. (2022). Nanotechnology: An approach to overcome bioavailability challenges of nutraceuticals. Journal of Drug Delivery Science and Technology, 72, Article 103418. https://doi.org/10.1016/j.jddst.2022.103418 Marena, G. D., Ramos, M. A. dos S., Carvalho, G. C., de Lima, L. C., Nascimento, A. L. C. S. do, Sábio, R. M., Rodero, C. F., Spósito, L., Bauab, T. M., & Chorilli, M. (2022). Development and characterization of an amphotericin B - loaded nanoemulsion applied to Candida auris biofilms control. Journal of Drug Delivery Science and Technology, 74, Article 103566. https://doi.org/10.1016/j.jddst.2022.103566 Marena, G. D., Ramos, M. A. dos S., Carvalho, G. C., Junior, J. A. P., Resende, F. A., Corrêa, I., Ono, G. Y. B., Sousa Araujo, V. H., de Camargo, B. A. F., Bauab, T. M., & Chorilli, M. (2022). Natural product‐based nanomedicine applied to fungal infection treatment: A review of the last 4 years. Phytotherapy Research, 36(7), 2710–2745. https://doi.org/10.1002/ptr.7460 Martins, F., Morgado, D. L., Sarmento, B., de Camargo, E. R., & das Neves, J. (2023). Chitosan-based sponges containing clotrimazole for the topical management of vulvovaginal candidiasis. International Journal of Pharmaceutics, 647, Article 123508. https://doi.org/10.1016/j.ijpharm.2023.123508 Martins, J. P., & Santos, H. A. (2023). Microfluidics as a Tool for the Synthesis of Advanced Drug Delivery Systems. In Lamprou, D. (Ed.), Nano- and Microfabrication Techniques in Drug Delivery (pp. 321–364). Springer. https://doi.org/10.1007/978-3-031-26908-0_13 Mba, I. E., & Nweze, E. I. (2020). Mechanism of Candida pathogenesis: revisiting the vital drivers. European Journal of Clinical Microbiology & Infectious Diseases, 39(10), 1797–1819. https://doi.org/10.1007/s10096-020-03912-w Mesini, A., Saffioti, C., Mariani, M., Florio, A., Medici, C., Moscatelli, A., & Castagnola, E. (2021). First Case of Candida auris Colonization in a Preterm, Extremely Low-Birth-Weight Newborn after Vaginal Delivery. Journal of Fungi, 7(8), Article 649. https://doi.org/10.3390/jof7080649 18 19 1 Introduction and aim of the thesis 20 Miguel Sábio, R., Corrêa Carvalho, G., Li, J., Chorilli, M., & Santos, H. A. (2023). Advanced porous materials for antimicrobial treatment. Nano Select, 1-20. https://doi.org/10.1002/nano.202300114 Ministério da Saúde. (2022). Protocolo clínico e diretrizes terapêuticas para atenção integral às pessoas com infecções sexualmente transmissíveis (IST). Ministério da Saúde. https://bvsms.saude.gov.br/bvs/publicacoes/protocolo_clinico_atecao_integral_ist.pdf Nsairat, H., Khater, D., Sayed, U., Odeh, F., Al Bawab, A., & Alshaer, W. (2022). Liposomes: structure, composition, types, and clinical applications. Heliyon, 8(5), Article e09394. https://doi.org/10.1016/j.heliyon.2022.e09394 Obeid, M. A., Alsaadi, M., & Aljabali, A. A. (2023). Recent updates in curcumin delivery. Journal of Liposome Research, 33(1), 53–64. https://doi.org/10.1080/08982104.2022.2086567 Parra‐Nieto, J., del Cid, M. A. G., Cárcer, I. A., & Baeza, A. (2021). Inorganic Porous Nanoparticles for Drug Delivery in Antitumoral Therapy. Biotechnology Journal, 16(2), Article 2000150. https://doi.org/10.1002/biot.202000150 Parsana, H., Chotaliya, M., & Dudhat, K. (2023). Formulation and Evaluation of Itraconazole Novel Nanosuspension-Based In Situ Gelling System for Vaginal Candidiasis Using 24 Factorial Design. BioNanoScience, 13(4), 1870–1884. https://doi.org/10.1007/s12668-023-01169-z Peters, B. M., Yano, J., Noverr, M. C., & Fidel, P. L. (2014). Candida Vaginitis: When Opportunism Knocks, the Host Responds. PLoS Pathogens, 10(4), Article e1003965. https://doi.org/10.1371/journal.ppat.1003965 Pongchaikul, P., Hajidariyor, T., Khetlai, N., Yu, Y.-S., Arjfuk, P., Khemthong, P., Wanmolee, W., Posoknistakul, P., Laosiripojana, N., Wu, K. C.-W., & Sakdaronnarong, C. (2023). Nanostructured N/S doped carbon dots/mesoporous silica nanoparticles and PVA composite hydrogel fabrication for anti-microbial and anti-biofilm application. International Journal of Pharmaceutics: X, 6, Article 100209. https://doi.org/10.1016/j.ijpx.2023.100209 Prem, P. N., Balu, K. K., Gandhi, S., & Kurian, G. A. (2023). Preparation of fisetin loaded mesoporous silica nanocarrier to attenuate ischemia reperfusion injury. Journal of Materials Research, 38(19), 4441–4453. https://doi.org/10.1557/s43578-023-01157-9 Sato, M. R., Oshiro-Junior, J. A., Rodero, C. F., Boni, F. I., Araújo, V. H. S., Bauab, T. M., Nicholas, D., Callan, J. F., & Chorilli, M. (2023). Enhancing Antifungal Treatment of Candida albicans with Hypericin-Loaded Nanostructured Lipid Carriers in Hydrogels: Characterization, In Vitro, and In Vivo Photodynamic Evaluation. Pharmaceuticals, 16(8), Article 1094. https://doi.org/10.3390/ph16081094 Seck, M. C., Engo, P. E., Gueye, P. A. T., Faye, C., Mbow, M., Diongue, K., Diallo, M. A., Ndiaye, M., Badiane, A. S., & others. (2023). Identification and antifungal susceptibility of Candida species isolated from vulvovaginal candidiasis in Dakar. Journal of Yeast and Fungal Research, 14(1), 1–7. Sharifi-Rad, J., Rayess, Y. El, Rizk, A. A., Sadaka, C., Zgheib, R., Zam, W., Sestito, S., Rapposelli, S., & others. (2020). Turmeric and Its Major Compound Curcumin on Health: Bioactive Effects and Safety Profiles for Food, Pharmaceutical, Biotechnological and Medicinal Applications. Frontiers in Pharmacology, 11, Article 01021. https://doi.org/10.3389/fphar.2020.01021 Sobel, J. D. (2023a). New Antifungals for Vulvovaginal Candidiasis: What Is Their Role? Clinical Infectious Diseases, 76(5), 783–785. https://doi.org/10.1093/cid/ciad002 Sobel, J. D. (2023b). Role of Antifungal Susceptibility Tests in the Treatment of Vulvovaginal Candidiasis. Current Infectious Disease Reports, 25(3), 29–32. https://doi.org/10.1007/s11908-023-00797-6 Sobel, R., Nyirjesy, P., Ghannoum, M., Delchev, D., Azie, N., Angulo, D., Harriott, I., Borroto‐Esoda, K., & Sobel, J. (2022). Efficacy and safety of oral ibrexafungerp for the treatment of acute vulvovaginal candidiasis: a global phase 3, randomised, placebo‐controlled superiority study (VANISH 306). BJOG: An International Journal of Obstetrics & Gynaecology, 129(3), 412–420. https://doi.org/10.1111/1471-0528.16972 Su, R., Li, P., Zhang, Y., Lv, Y., Wen, F., & Su, W. (2023). Polydopamine/tannic acid/chitosan/poloxamer 407/188 thermosensitive hydrogel for antibacterial and wound healing. Carbohydrate Polymers, 302, Article 120349. https://doi.org/10.1016/j.carbpol.2022.120349 Sukmawaty, M., Sartini, Permana, A. D., Mudjahid, M., Roska, T. P., & Rahman, L. (2023). Lipid Complex-Itraconazole in Thermosensitive Mucoadhesive Vaginal Gel to Enhance the Effectiveness of Therapy for Vulvovaginal Candidiasis: Formulation, Optimization, Characterization, and Ex vivo Evaluation. Journal of Pharmaceutical Innovation, 18, 1546-1559. https://doi.org/10.1007/s12247-023-09738-1 Sun, Z., Ge, X., Qiu, B., Xiang, Z., Jiang, C., Wu, J., & Li, Y. (2023). Vulvovaginal candidiasis and vaginal microflora interaction: Microflora changes and probiotic therapy. Frontiers in Cellular and Infection Microbiology, 13, Article 1123026. https://doi.org/10.3389/fcimb.2023.1123026 WHO. (2022). WHO fungal priority pathogens list to guide research, development and public health action (World Health Organization (WHO). https://www.who.int/publications/i/item/9789240060241 Wu, L., Gou, K., Guo, X., Guo, Y., Chen, M., Hou, J., Li, S., & Li, H. (2021). Dual response to pH and chiral microenvironments for the release of a flurbiprofen-loaded chiral self-assembled mesoporous silica drug delivery system. Colloids and Surfaces B: Biointerfaces, 199, Article 111501. https://doi.org/10.1016/j.colsurfb.2020.111501 Yang, H., Cui, X., Li, S., Cen, Y., Deng, T., Wang, J., Olsbye, U., & Fan, W. (2020). Developing a general method for encapsulation of metal oxide nanoparticles in mesoporous silica shell by unraveling its formation mechanism. Microporous and Mesoporous Materials, 305, Article 110381. https://doi.org/10.1016/j.micromeso.2020.110381 Żyrek, D., Wajda, A., Czechowicz, P., Nowicka, J., Jaśkiewicz, M., Neubauer, D., & Kamysz, W. (2021). The Antimicrobial Activity of Omiganan Alone and In Combination against Candida Isolated from Vulvovaginal Candidiasis and Bloodstream Infections. Antibiotics, 10(8), Article 1001. https://doi.org/10.3390/antibiotics10081001 20 Chapter 1 20 Miguel Sábio, R., Corrêa Carvalho, G., Li, J., Chorilli, M., & Santos, H. A. (2023). Advanced porous materials for antimicrobial treatment. Nano Select, 1-20. https://doi.org/10.1002/nano.202300114 Ministério da Saúde. (2022). Protocolo clínico e diretrizes terapêuticas para atenção integral às pessoas com infecções sexualmente transmissíveis (IST). Ministério da Saúde. https://bvsms.saude.gov.br/bvs/publicacoes/protocolo_clinico_atecao_integral_ist.pdf Nsairat, H., Khater, D., Sayed, U., Odeh, F., Al Bawab, A., & Alshaer, W. (2022). Liposomes: structure, composition, types, and clinical applications. Heliyon, 8(5), Article e09394. https://doi.org/10.1016/j.heliyon.2022.e09394 Obeid, M. A., Alsaadi, M., & Aljabali, A. A. (2023). Recent updates in curcumin delivery. Journal of Liposome Research, 33(1), 53–64. https://doi.org/10.1080/08982104.2022.2086567 Parra‐Nieto, J., del Cid, M. A. G., Cárcer, I. A., & Baeza, A. (2021). Inorganic Porous Nanoparticles for Drug Delivery in Antitumoral Therapy. Biotechnology Journal, 16(2), Article 2000150. https://doi.org/10.1002/biot.202000150 Parsana, H., Chotaliya, M., & Dudhat, K. (2023). Formulation and Evaluation of Itraconazole Novel Nanosuspension-Based In Situ Gelling System for Vaginal Candidiasis Using 24 Factorial Design. BioNanoScience, 13(4), 1870–1884. https://doi.org/10.1007/s12668-023-01169-z Peters, B. M., Yano, J., Noverr, M. C., & Fidel, P. L. (2014). Candida Vaginitis: When Opportunism Knocks, the Host Responds. PLoS Pathogens, 10(4), Article e1003965. https://doi.org/10.1371/journal.ppat.1003965 Pongchaikul, P., Hajidariyor, T., Khetlai, N., Yu, Y.-S., Arjfuk, P., Khemthong, P., Wanmolee, W., Posoknistakul, P., Laosiripojana, N., Wu, K. C.-W., & Sakdaronnarong, C. (2023). Nanostructured N/S doped carbon dots/mesoporous silica nanoparticles and PVA composite hydrogel fabrication for anti-microbial and anti-biofilm application. International Journal of Pharmaceutics: X, 6, Article 100209. https://doi.org/10.1016/j.ijpx.2023.100209 Prem, P. N., Balu, K. K., Gandhi, S., & Kurian, G. A. (2023). Preparation of fisetin loaded mesoporous silica nanocarrier to attenuate ischemia reperfusion injury. Journal of Materials Research, 38(19), 4441–4453. https://doi.org/10.1557/s43578-023-01157-9 Sato, M. R., Oshiro-Junior, J. A., Rodero, C. F., Boni, F. I., Araújo, V. H. S., Bauab, T. M., Nicholas, D., Callan, J. F., & Chorilli, M. (2023). Enhancing Antifungal Treatment of Candida albicans with Hypericin-Loaded Nanostructured Lipid Carriers in Hydrogels: Characterization, In Vitro, and In Vivo Photodynamic Evaluation. Pharmaceuticals, 16(8), Article 1094. https://doi.org/10.3390/ph16081094 Seck, M. C., Engo, P. E., Gueye, P. A. T., Faye, C., Mbow, M., Diongue, K., Diallo, M. A., Ndiaye, M., Badiane, A. S., & others. (2023). Identification and antifungal susceptibility of Candida species isolated from vulvovaginal candidiasis in Dakar. Journal of Yeast and Fungal Research, 14(1), 1–7. Sharifi-Rad, J., Rayess, Y. El, Rizk, A. A., Sadaka, C., Zgheib, R., Zam, W., Sestito, S., Rapposelli, S., & others. (2020). Turmeric and Its Major Compound Curcumin on Health: Bioactive Effects and Safety Profiles for Food, Pharmaceutical, Biotechnological and Medicinal Applications. Frontiers in Pharmacology, 11, Article 01021. https://doi.org/10.3389/fphar.2020.01021 Sobel, J. D. (2023a). New Antifungals for Vulvovaginal Candidiasis: What Is Their Role? Clinical Infectious Diseases, 76(5), 783–785. https://doi.org/10.1093/cid/ciad002 Sobel, J. D. (2023b). Role of Antifungal Susceptibility Tests in the Treatment of Vulvovaginal Candidiasis. Current Infectious Disease Reports, 25(3), 29–32. https://doi.org/10.1007/s11908-023-00797-6 Sobel, R., Nyirjesy, P., Ghannoum, M., Delchev, D., Azie, N., Angulo, D., Harriott, I., Borroto‐Esoda, K., & Sobel, J. (2022). Efficacy and safety of oral ibrexafungerp for the treatment of acute vulvovaginal candidiasis: a global phase 3, randomised, placebo‐controlled superiority study (VANISH 306). BJOG: An International Journal of Obstetrics & Gynaecology, 129(3), 412–420. https://doi.org/10.1111/1471-0528.16972 Su, R., Li, P., Zhang, Y., Lv, Y., Wen, F., & Su, W. (2023). Polydopamine/tannic acid/chitosan/poloxamer 407/188 thermosensitive hydrogel for antibacterial and wound healing. Carbohydrate Polymers, 302, Article 120349. https://doi.org/10.1016/j.carbpol.2022.120349 Sukmawaty, M., Sartini, Permana, A. D., Mudjahid, M., Roska, T. P., & Rahman, L. (2023). Lipid Complex-Itraconazole in Thermosensitive Mucoadhesive Vaginal Gel to Enhance the Effectiveness of Therapy for Vulvovaginal Candidiasis: Formulation, Optimization, Characterization, and Ex vivo Evaluation. Journal of Pharmaceutical Innovation, 18, 1546-1559. https://doi.org/10.1007/s12247-023-09738-1 Sun, Z., Ge, X., Qiu, B., Xiang, Z., Jiang, C., Wu, J., & Li, Y. (2023). Vulvovaginal candidiasis and vaginal microflora interaction: Microflora changes and probiotic therapy. Frontiers in Cellular and Infection Microbiology, 13, Article 1123026. https://doi.org/10.3389/fcimb.2023.1123026 WHO. (2022). WHO fungal priority pathogens list to guide research, development and public health action (World Health Organization (WHO). https://www.who.int/publications/i/item/9789240060241 Wu, L., Gou, K., Guo, X., Guo, Y., Chen, M., Hou, J., Li, S., & Li, H. (2021). Dual response to pH and chiral microenvironments for the release of a flurbiprofen-loaded chiral self-assembled mesoporous silica drug delivery system. Colloids and Surfaces B: Biointerfaces, 199, Article 111501. https://doi.org/10.1016/j.colsurfb.2020.111501 Yang, H., Cui, X., Li, S., Cen, Y., Deng, T., Wang, J., Olsbye, U., & Fan, W. (2020). Developing a general method for encapsulation of metal oxide nanoparticles in mesoporous silica shell by unraveling its formation mechanism. Microporous and Mesoporous Materials, 305, Article 110381. https://doi.org/10.1016/j.micromeso.2020.110381 Żyrek, D., Wajda, A., Czechowicz, P., Nowicka, J., Jaśkiewicz, M., Neubauer, D., & Kamysz, W. (2021). The Antimicrobial Activity of Omiganan Alone and In Combination against Candida Isolated from Vulvovaginal Candidiasis and Bloodstream Infections. Antibiotics, 10(8), Article 1001. https://doi.org/10.3390/antibiotics10081001 21 CHAPTER 2 Prevalence of vulvovaginal candidiasis in Brazil: A systematic review Gabriela Corrêa Carvalho, Rafaela Aparecida Prata de Oliveira, Victor Hugo Sousa Araújo, Rafael Miguel Sábio, Lídia Raquel de Carvalho, Taís Maria Bauab, Ione Corrêa, Marlus Chorilli* Medical Mycology, v. 59, n. 10, pp. 946-957, 2021. Reprinted with permission from Oxford University Press 20 21 22 Abstract Vulvovaginal candidiasis (CVV) is a condition whose signs and symptoms are related to inflammation caused by Candida spp infection. It is the second leading cause of vaginitis in the world, representing a public health problem. The present systematic review comes with the proposal of analyze and identify the available evidence on CVV prevalence in Brazil, pointing out its variability by regions. For this, a systematic literature review was carried out with meta-analysis of cross-sectional and cohort studies, following the Preferred Reporting Items for Systematic Reviews and Meta- Analyzes (PRISMA) guide recommendations, and was registered in the International Prospective Register of Systematic Reviews (PROSPERO 2020 CRD42020181695). The databases used for survey were LILACS, Scielo, Scopus, PUBMED, Web of Science and CINAHL. Fifteen studies were selected to estimate CVV prevalence in the Brazilian territory. South and Southeast regions have higher prevalences than the North and Northeast regions, no data were found for the Midwest region. The estimated prevalence for Brazil is 18%, however, it is suggested that this number is higher due to underreporting and the presence of asymptomatic cases. Therefore, new epidemiological studies are recommended throughout Brazil, to elucidate the profile of this disease in the country, in addition to assisting in the elaboration of an appropriate prevention plan by state. 22 Chapter 2 22 Abstract Vulvovaginal candidiasis (CVV) is a condition whose signs and symptoms are related to inflammation caused by Candida spp infection. It is the second leading cause of vaginitis in the world, representing a public health problem. The present systematic review comes with the proposal of analyze and identify the available evidence on CVV prevalence in Brazil, pointing out its variability by regions. For this, a systematic literature review was carried out with meta-analysis of cross-sectional and cohort studies, following the Preferred Reporting Items for Systematic Reviews and Meta- Analyzes (PRISMA) guide recommendations, and was registered in the International Prospective Register of Systematic Reviews (PROSPERO 2020 CRD42020181695). The databases used for survey were LILACS, Scielo, Scopus, PUBMED, Web of Science and CINAHL. Fifteen studies were selected to estimate CVV prevalence in the Brazilian territory. South and Southeast regions have higher prevalences than the North and Northeast regions, no data were found for the Midwest region. The estimated prevalence for Brazil is 18%, however, it is suggested that this number is higher due to underreporting and the presence of asymptomatic cases. Therefore, new epidemiological studies are recommended throughout Brazil, to elucidate the profile of this disease in the country, in addition to assisting in the elaboration of an appropriate prevention plan by state. 23 1. Introduction Vulvovaginal Candidiasis (VVC) is a disease which signs and symptoms are related to inflammation resulting from infection by Candida spp. (Bonifácio et al., 2015; dos Santos Ramos et al., 2016; Sangamithra et al., 2013). After bacterial vaginosis, VVC is the most common cause of vaginitis and it is estimated that about 70-75% of women will present this pathology at some point in their lives (Anderson et al., 1989; Donders & Sobel, 2017; Sobel, 2007). According to Gonçalves and collaborators (2016), the incidence of VVC in different countries varies from 12.1% to 57.3%, where Brazil has the third highest index, behind only Nigeria and Tunisia (Amouri et al., 2011; Okungbowa et al., 2003). These data are reinforced by several other studies carried out in other countries such as Burkina Faso (Sangaré et al., 2018), Argentina (Mucci et al., 2017), Lebanon (Ghaddar et al., 2020), India (Krishnasamy et al., 2020), Saudi Arabia (Venugopal et al., 2020), Ghana (Konadu et al., 2019; Waikhom et al., 2020) and India (RANI et al., 2020), where the prevalence values obtained were within this range. Despite VVC prevalence, in Brazil there is a lack and an out datedness of epidemiological data on this disease, what leads to VVC underestimation (Brandão et al., 2018). Which can be attributed not only to the fact that it is not a notifiable disease, but also due its asymptomatic cases (Brandão et al., 2018). This lack of information can be detected in other countries like India (Rani et al., 2020), Ethiopia (Tufa & Denning, 2019), Namibia (Dunaiski & Denning, 2019) Saudi Arabia (Venugopal et al., 2020) and Greece (Gamaletsou et al., 2016), where this lack of data motivated epidemiological studies to be carried out. In a study conducted in Mumbai, India, it was observed that 30.7% of women without VVC symptoms had Candida sp. in their vaginal microbiota (Pramanick et al., 2019). Although the prevalence of this disease varies according to the different regions (Farr et al., 2021) a similar value, 33.83%, was also observed by work carried out by researchers from Turkey on asymptomatic women (Cengiz et al., 2020). It also stands out that as it is a local infection, not associated with mortality, patients sometimes do not look for a health service, leading to a self-medication, this fact also corroborates with underestimation data (Czechowicz et al., 2021; Venugopal et al., 2020; Yano et al., 2019). In a study conducted in the United States of America, 24.1% of women were self-diagnosed with VVC, which is worrying when thinking about self-medication and resistance to antifungals (Araujo et al., 2020; Yano et al., 2019). Due to the fact that the data found in the literature regarding the epidemiological profile of VVC in Brazil are obsolete and incomplete, a new assessment about the behavior of this pathology is necessary. Considering the previous aspects, the present systematic review comes with the proposal to analyze and identify the available evidence on the prevalence of VVC in Brazil, pointing out its 22 23 2 Prevalence of vulvovaginal candidiasis in Brazil 24 variability in different regions and updating the data related to the epidemiological profile to assist in a better understanding about this pathology in the Brazilian territory. 2. Methodology 2.1. Protocol design and registration A systematic literature review was carried out with a meta-analysis of cross-sectional and cohort studies. The study protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO) under the identification: PR