UNIVERSIDADE ESTADUAL PAULISTA "JÚLIO DE MESQUITA FILHO" Câmpus Bauru Leonardo Francisco Gonçalves Dias Immobilization of bisphosphonates on nanostructured films of TiO2 and hydroxyapatite Bauru 2022 Leonardo Francisco Gonçalves Dias Immobilization of bisphosphonates on nanostructured films of TiO2 and hydroxyapatite Tese apresentada como requisito à obtenção do título de Doutor à Universidade Estadual Paulista “Júlio de Mesquita Filho” – Programa de Pós-graduação em Ciência e Tecnologia de Materiais, sob orientação do Prof. Dr. Paulo Noronha Lisboa-Filho Financiadora: FAPESP - Proc. 2018/07520-3 Orientador: Prof. Dr. Paulo Noronha Lisboa- Filho Coorientadora: Drª. Erika Soares Bronze-Uhle Faculdade de Ciências Bauru 2022 Dias, Leonardo Francisco Gonçalves. Immobilization of bisphosphonates on nanostructured films of TiO2 and hy- droxyapatite / Leonardo Francisco Gonçalves Dias. – Bauru, 2022 135 f. : il., tabs. Supervisor: Prof. Dr. Paulo Noronha Lisboa-Filho Tese (doutorado) - Universidade Estadual Paulista “Júlio de Mesquita Filho", Faculdade de Ciências. 1. bisphosphonates. 2. titanium dioxide. 3. hydroxyapatite. I. Lisboa-Filho, Paulo Noronha II. Universidade Estadual Paulista “Júlio de Mesquita Filho", Faculdade de Ciências. III. Título. CDU – 518.72:76 Leonardo Francisco Gonçalves Dias Immobilization of bisphosphonates on nanostructured films of TiO2 and hydroxyapatite Tese apresentada como requisito à obtenção do título de Doutor à Universidade Estadual Paulista “Júlio de Mesquita Filho” – Programa de Pós-graduação em Ciência e Tecnologia de Materiais, sob orientação do Prof. Dr. Paulo Noronha Lisboa-Filho Financiadora: FAPESP - Proc. 2018/07520-3 Bauru 31 de outubro de 2022 Dedico este trabalho aos meus pais Israel e Valda, minha esposa Marina e minha filha Mariana. Agradecimentos Gostaria de agradecer à minha companheira e esposa Marina, a pessoa que esteve mais próxima de mim durante toda essa caminhanda. Aos momentos de conversa, carinho e acolhimento que você sempre proporcionou. Ao seu lado sinto que sou capaz de fazer tudo. Aos meus pais, Israel e Valda por desde sempre me apoairem a seguir os meus sonhos, mesmo as vezes não os entendendo completamente. O apoio de vocês não só para este trabalho, mas para tudo o que fiz e farei é fundamental para mim. À minha filha Mariana, que com seu jeito doce e empático de ver a vida sempre fez com que eu me sentisse especial. À minha amiga Maithe por sempre acreditar nas minhas capacidades. À minha terapeuta Bárbara Campos pelu auxílio fundamental nessa jornada. Aos meus orientadores Prof. Dr. Paulo Noronha e Drª. Erika Soares pelos ensina- mentos não só sobre ciência de materiais como também sobre a vida e vinhos, além dos incontáveis cafés. Vocês sempre me apoiaram, mesmo na época em que eu era apenas um calouro na universidade e me acolheram com muito carinho nos momentos de dificuldade. Aos meus amigos de laboratório pelas conversas e apoio. Por último, mas não menos importante aos técnicos e servidores da universidade. Ao programa de pós-graduação em ciência e tecnologia de materiais, a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), a Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP pelo financiamento através dos projetos 2018/07520-3 e 2019/13100-0. Pouca saúde, muita saúva, os males do Brasil são. (Macunaíma - Mário de Andrade) Abstract Bisphosphonates are a class of organic molecules used for bone growth, however, the prolonged use of these molecules is related to mandibular osteonecrosis. To maintain the ability to promote bone growth, the immobilization of these molecules on the surface of implants is an alternative to increasing the biocompatibility of implants without presenting the side effects of prolonged exposu re. This thesis aims to collaborate with data and discussions of bisphosphonates immobilized on TiO2 surfaces of hydroxyapatite (HA), based mainly on measurements of X-ray photoelectron spectroscopy, zeta potential, and atomic force microscopy. First, layers of octadecylphosphonic acid on TiO2 obtained by sputtering were investigated, then bisphosphonates on different surfaces, such as TiO2 by sol-gel and hydroxyapatite. Data obtained from computer simulation were used and discussed together with the experimental data. The results indicate that bisphosphonates adsorb on the surface exposing the phosphonate groups, and promoting a hydrophilic surface. Furthermore, changes in pH alter the surface charge due to the presence of P-OH and -N+ species. On titanium dioxide surfaces, adsorption can be enhanced by the hydroxylation process, as shown in the adsorption curves of bisphosphonates. No cytotoxic effec was observed on prepared samples. The applied strategy is a suitable strategy for the investigation of bioactive molecules and can be used as a basis for other compounds. Keywords: bisphosphonate, titanium dioxide, hydroxyapatite, surface characterization. Resumo Os bisfosfonatos são uma classe de moléculas orgânicas utilizadas para o crescimento ósseo, porém, o uso prolongado desses dessas moléculas está relacionado à osteonecrose mandibular. Para manter a capacidade de promover o crescimento ósseo, a imobilização dessas moléculas na superfície dos implantes é uma alternativa para aumentar a biocompa- tibilidade dos implantes sem apresentar os efeitos colaterais da exposição prolongada a essas moléculas. Esta tese visa colaborar com dados e discussões de bifosfonatos imobili- zados em superfícies de TiO2 de hidroxiapatita (HA), com base principalmente em medidas de espectroscopia de fotoelétrons excitados por raios X, potencial zeta e microscopia de força atômica. Primeiramente, foram investigadas camadas de ácido octadecilfosfônico sobre TiO2 obtidas por sputtering, depois bifosfonatos em diferentes superfícies, como TiO2 por sol-gel e hidroxiapatita. Os dados obtidos a partir de simulação computacional foram utilizados e discutidos juntamente com os dados experimentais. Os resultados indicam que os bisfosfonatos adsorvem na superfície expondo os grupos fosfonatos e promovendo uma superfície hidrofílica. Além disso, mudanças no pH alteram a carga superficial devido à presença de espécies P-OH e -N+. Em superfícies de dióxido de titânio, a adsorção pode ser potencializada pelo processo de hidroxilação, como mostrado nas curvas de adsorção dos bifosfonatos. Nenhum efeito citotóxico foi observado nas amostras preparadas. A estratégia aplicada é se mostrou adequada para a investigação de moléculas bioativas e pode ser utilizada como base para outros compostos. Palavras-chaves: bisfosfonato, dióxido de titânio, hidroxiapatita, caracterização de superfí- cies. Resumo expandido O tecido ósseo é um dos principais componentes do esqueleto humano. Entre as suas funções, destacam-se a proteção dos órgãos vitais, suporte para os músculos, além de ser um reservatório de íons de cálcio e fósforo. A manutenção adequada desse tecido depende do equilíbrio da atividade de células chamadas osteoblastos e osteoclastos. Os osteoblastos estão diretamente relacionados com crescimento e renovação óssea, enquanto os oste- oclastos são responsáveis por reabsorver o tecido ósseo. Um desequilíbrio da atividade dessas células pode resultar em perda óssea, característica comum da osteoporose. Outro fator que pode causar um desequilíbrio são traumas causados em acidentes de trânsito, por exemplo. Em alguns casos é necessária a substituição total ou parcial de alguns ossos, que em geral são substituídos por implantes metálicos, compostos principalmente por titânio. Ao ser colocado dentro do organismo, um implante sofre uma reação de corpo estranho que em casos graves pode levar a sua rejeição completa, sendo necessário novos procedimentos cirúrgicos. Com o objetivo de reduzir as falhas dos implantes e/ou acelerar a integração entre o tecido vivo e o implante, pesquisadores têm se dedicado a modificar a superfície desses materiais para atingir esse objetivo. Dentre as modificações de superfície destacam-se a deposição de filmes finos e a funcio- nalização. A primeira opção consiste na deposição de finas camadas de outros materiais como por exemplo o dióxido de titânio, hidroxiapatita e compósitos dos dois materiais. O TiO2 devido à presença de hidroxilas em sua superfície favorece adsorção de proteínas que auxiliam no processo de integração tecido-implante. A hidroxiapatita, devido a sua presença no tecido ósseo é um dos recobrimentos que maior favorece a biocompatibilidade, entretanto possui uma baixa adesão em superfícies metálicas. O compósito dos dois materi- ais alia a biocompatibilidade com adesão implante-filme. O segundo processo, chamado funcionalização consiste na adição de grupos funcionais sobre a superfícies. Dentre as moléculas utilizadas para isso destacam-se os bisfosfonatos. Bisfosfonatos são uma família de moléculas orgânicas análogos ao pirofosfato, inibidores de mineralização, que são utilizadas principalmente para o tratamento de problemas ósseos como osteoporose e doença de Paget, promovendo principalmente um crescimento ósseo além da diminuição da atividade dos osteoclastos. Entretanto, o uso contínuo desse com- posto está associado a osteonecrose mandibular. Dessa forma o uso dessas moléculas como modificadores de superfície, pode aliar a capacidade de crescimento ósseo reduzindo os efeitos indesejados. Diante disso, a presente tese tem como objetivo contribuir com dados e discussões sobre su- perfícies de TiO2, HA e TiO2+HA, recobertas com bisfosfonatos, especificamente etidronato, alendronato e risedronato. Para isso técnicas como espectroscopia de fotoelétrons de raio-X foram amplamente utilizadas, bem como potencial zeta, ângulo de contato e microscopia de força atômica. Os resultados deste trabalho foram apresentados em quatro capítulos em formato de artigos. A primeira publicação intitulada "New details of assembling bioactive films from dispersions of amphiphilic molecules on titania surfaces", consiste na intestigação de filmes finos de um silano e um fosfonato, respectivamente, cloreto de dimetiloctadecil (3-trimetoxissililpropil) amônio (DMOAP) e ácido octadecilfosfônico (ODPA), sobre dióxido de titânio. As inves- tigações mostraram uma relação entre a estrutura dos filmes e a atividade bactericida, especialmente para o DMOAP. Os resultados de potencial zeta juntamente com espectros- copia de fotoelétrons de raio-X demostraram que as mudanças na carga superficial de filmes de ODPA ocorre devido à presença de grupos P-OH não ligados. Os resultados obtidos, principalmente para as monocamadas de ODPA servem como base para a interpretação e discussão de dados envolvendo bisfosfonatos. A segunda publicação nomeada "Bisphosphonates on smooth TiO2: modeling and cha- racterization", utiliza principalmente dados de simulação computacional, potencial zeta e espectroscopia de fotoelétrons de raio-X para discutir as características físico-químicas de filmes de etidronato, alendronato e risedronato sobre dióxido de titânio. Os resultados demonstram mais uma vez a influência de grupos P-OH na superfície, bem como do alen- dronato e risedronato, que contêm nitrogênio. Além disso, artigo indica que os bisfosfonatos adsorvem na superfície através da condensação das hidroxilas do TiO2 e dos grupos P-OH, formando ligações do tipo Ti-O-P. Bisfosfonatos que contêm nitrogênio apresentam ligações de hidrogênio entre as hidroxilas e os grupos nitrogenados. A terceira publicação, "Adsorption and X-ray photoelectron spectroscopy investigation of bisphosphonates on titania and hydroxyapatite surfaces", apresenta a curva de adsorção dos bisfosfonatos em TiO2 junto com uma investigação da adsorção dessas moléculas em TiO2, hidroxiapatita e superfícies mistas. Os resultados mostraram que a hidroxilação do TiO2 aumenta a quantidade de bisfosfonato adsorvida, esse efeito foi atribuído a um aumento da deprotonação das moléculas causas pelos grupos OH terminais. Os resultados de espectroscopia de fotoelétrons de raios-X mostram uma adsorção competitiva entre os bisfosfonatos e grupos carboxilas, que é menor em superfícies contendo hidroxiapatita. Por fim observou-se que as moléculas adsorvem no TiO2 seguindo a topografia da superfíce enquanto na hidroxiapatita os bisfosfonatos acumulam ao redor dos íons de cálcio, isso se reflete em um aumento da rugosidade apenas para a superfície de hidroxiapatita. O Capítulo 6 apresenta os resultados microbiológicos obtidos até a conclusão dessa tese. Os resultados indicam uma tendência de superfícies contendo risedronato em limitar o crescimento de S. aureus, essa tendência foi associdada a um efeito sinergético entre a camada adsorvida e a rugosidade do material. Por fim, não foram observados efeitos citotóxicos em células mesenquimais ósseas humanas. As publicações apresentadas estão de acordo com os objetivos propostos nessa tese. A estratégia proposta mostrou-se eficaz para evidenciar os aspectos da adsorção dos bisfosfonatos, bem como as propriedades de suas camadas. A estratégia aqui apresentada pode ser aplicada em outras moléculas bioativas. Palavras-chaves: bisfosfonato, dióxido de titânio, hidroxiapatita, caracterização de superfí- cies. List of Figures Figure 1 – Schematic representation of mineralization in bone matrix . . . . . . . . 23 Figure 2 – Synthesis mechanism of titanium dioxide by sol-gel route using titanium isopropoxide as precursor. . . . . . . . . . . . . . . . . . . . . . . . . . 25 Figure 3 – a) General structure of bisphosphonates, b) etidronate, c) alendronate and d) risedronate linear structure. . . . . . . . . . . . . . . . . . . . . . 26 Figure 4 – Effects of BPs on bone metabolism. A) Transformation of non-N-BPs into cytotoxic ATP analogs prompts osteoclast apoptosis, while N-BPs induce osteoclast apoptosis by inhibiting FPPS. B) BPs modulate the expression of osteoclast-related genes and promote the expression of osteogenicrelated genes. C) BPs restrain BMSC adipogenic differentiation and promote their osteogenic differentiation. D) BPs inhibit apoptosis of osteoblasts and osteocytes. . . . . . . . . . . . . . . . . . . . . . . . . 28 Figure 5 – Linear structure of (a) DMOAP and (b) ODPA. . . . . . . . . . . . . . . . 33 Figure 6 – XPS high resolution spectra for investigated bulk sample ODPA, (a) C1s, (b) O1s and (c) P2p signal. . . . . . . . . . . . . . . . . . . . . . . . . . 42 Figure 7 – XPS C1s high-resolution spectra for DMOAP, (a) bulk, (b) water exposed, and (c) heated blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Figure 8 – XPS high-resolution spectra for sputtered titanium dioxide, (a) Ti2p, (b) O1s, (c) C1s and (d) N1s signal. . . . . . . . . . . . . . . . . . . . . . . 46 Figure 9 – High-resolution C1s and O1s XPS spectra obtained for ODPA layers on titania after immersion in ODPA solution for 4 h (a) and (c); 24 h (b) and (d). 47 Figure 10 – XPS high-resolution spectra for DMOAP layer on titania, (a) C1s, (b) O1s, (c) N1s and (d) Si2p signals. . . . . . . . . . . . . . . . . . . . . . . . . 50 Figure 11 – Zeta potential results for the studied layers on smooth titanium dioxide. . 54 Figure 12 – AFM height and adhesion force image for the investigated layers. . . . . 55 Figure 13 – AFM height profile of (a) sputtered titanium dioxide and (b) ODPA monolayer. 56 Figure 14 – S. mutans growth after exposition to different samples during three differ- ent periods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Figure 15 – a) General structure of bisphosphonic acids and linear structure of b) etidronic acid, c) alendronic acid and d) risedronic acid. . . . . . . . . . 61 Figure 16 – Structure, Fukui indices, and map of electrostatic potential (MEP) of studied molecules when dissolved in water. The number of asterisks (*) in each molecule is used to indicate the scale of the map of the electrostatic potential, because the protonated/deprotonated states of the molecule are unbalance in the charge. . . . . . . . . . . . . . . . . . . . . . . . . 64 Figure 17 – C1s, O1s, and N1s high-resolution spectra for sputtered-deposited titania; C1s high-resolution spectra of etidronic acid (ETI), alendronic acid (ALE), and risedronic acid (RIS) layers adsorbed on titania. . . . . . . . . . . . 66 Figure 18 – O1s and N1s XPS signals for etidronic acid (ETI), alendronic acid (ALE), and risedronic (RIS) adsorbed on titania. . . . . . . . . . . . . . . . . . 68 Figure 19 – Representation of I) etidronic acid, II) alendronic acid, and III) risedronic acid molecules adsorbed on titania surface. Hidrogen atoms are rep- resented in white, carbon in grey, oxygen in red, nitrogen in blue, and phosphorous in yellow. . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Figure 20 – Zeta potential measurements of bisphosphonates adsorbed on TiO2. . . 72 Figure 21 – AFM images of bisphosphonates adsorbed on smooth titania. . . . . . . 73 Figure 22 – Linear structures of BPs, etidronate, alendronate, and risedronate. . . . 76 Figure 23 – Adsorption curves of BP adsorbed on titania particles. Dotted lines repre- sent theoretical curves. . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Figure 24 – XRD patterns of dried dispersions. . . . . . . . . . . . . . . . . . . . . . 82 Figure 25 – SEM images of prepared samples. . . . . . . . . . . . . . . . . . . . . . 83 Figure 26 – C 1s high-resolution XPS spectra of prepared films. BP (4 mM) was employed for functionalization. . . . . . . . . . . . . . . . . . . . . . . . 86 Figure 27 – O 1s high-resolution XPS spectra of prepared films. BP (4 mM) was employed for functionalization. . . . . . . . . . . . . . . . . . . . . . . . 87 Figure 28 – AFM images of prepared samples. BP (4 mM) was used for functionalization. 88 Figure 29 – In vitro antibacterial activity of bisphosphonate-based coatings. (A) Schematic representation of Staphylococcus aureus biofilm model used (B) Colony- forming units count (Log10 CFU/mL) of S. aureus after 24 h of biofilm formation. (b’) Percentage of bacterial reduction (% ) of treated surfaces to control. Data are expressed as mean ± standard deviation. Statisti- cally, significant differences are indicated as *p < 0.05 (HSD Tukey and Bonferroni t-test). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Figure 30 – Determination of the minimum inhibitory concentration of risedronate. (A) MIC of risedronate from 1 mg/mL to 0.001 mg/mL. (B) Agar diffusion test with S. aureus. NS = no statistically significant differences among the groups. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Figure 31 – MTT assay. Absorbance is expressed as a measure of cell metabolism for hBMSC cells cultured on control and bisphosphonate-based coatings for each deposition process (different colors) in 3 (A) and 7 (B) days of culture. *p < 0.05 (HSD Tukey and Bonferroni t-test). . . . . . . . . . . . 95 Figure 32 – High-resolution spectra a) Si 2p and b) P 2p for investigated bulk ODPA on a silicon wafer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Figure 33 – O 1s, N 1s, Si 2p and Cl 2p high-resolution spectra for the investigated bulk DMOAP samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Figure 34 – C1s and O1s high-resolution spectra for octadecylphosphonic acid ad- sorbed on titanium dioxide. . . . . . . . . . . . . . . . . . . . . . . . . . 121 Figure 35 – P2p high-resolution spectra for octadecylphosphonic acid adsorbed on titanium dioxide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Figure 36 – Si 2p high-resolution spectra for DMOAP adsorbed on titania. Where "A" represent addition of aqueous HCl on the solution and "N" is the solution without the addition of HCl. . . . . . . . . . . . . . . . . . . . . . . . . . 123 Figure 37 – MODA plot of the computational methods employed. . . . . . . . . . . . 125 Figure 38 – Characterization data (CHADA) documentation was applied for XPS mea- surements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Figure 39 – Structure, f+, f− and the MEP of all the structures simulated in vacuum. . 128 Figure 40 – Structure, f+, f− and the MEP of all the structures simulated in aqueous medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Figure 41 – C 1s high-resolution spectra of bulk etidronate. . . . . . . . . . . . . . . 130 Figure 42 – XRD of TiO2 particles. . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Figure 43 – Confocal images of prepared samples. . . . . . . . . . . . . . . . . . . 132 Figure 44 – Map of chemical composition of TiO2 film. . . . . . . . . . . . . . . . . . 132 Figure 45 – Map of chemical composition of TiO2:HA 1:1 film. . . . . . . . . . . . . . 133 Figure 46 – Map of chemical composition of TiO2:HA 1:2 film. . . . . . . . . . . . . . 134 Figure 47 – Map of chemical composition of HA film. . . . . . . . . . . . . . . . . . . 135 Figure 48 – N 1s high-resolution spectra of samples containing nitrogen. . . . . . . . 135 List of Tables Table 1 – Parameters of immersion, drying method, and sample name for DMOAP/titania films prepared from two different DMOAP solutions: DMOAP-N (without acid addition) and DMOAP-A (with acid addition) . . . . . . . . . . . . . . 38 Table 2 – Comparison between expected for homogeneous solid ODPA and obtained values from the XPS-based evaluations of atomic concentration . . . . . 42 Table 3 – Ratios between the atomic concentrations of carbon [C], phosphorous [P], and oxygen [O] species, as expected from stoichiometry and found in XPS investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Table 4 – Atomic concentration in at% of the distinct surface species by XPS investi- gations, compared to stoichiometrically expected values. . . . . . . . . . 43 Table 5 – Concentration ratios obtained from fitting C1s signals of DMOAP bulk samples prepared following different processes. . . . . . . . . . . . . . . 44 Table 6 – Normalized XPS signal intensity ratios for ODPA samples adsorbed on titania, based on the sample immersed for 24 h in ethanolic ODPA solution and then rinsed with ethanol (subsequently labelled “SAM”), and estimated layer thickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Table 7 – Atomic concentration in at% of the distinct surface species from XPS investigations of DMOAP/titania films . . . . . . . . . . . . . . . . . . . . 49 Table 8 – Normalized atomic concentration ratios for freshly prepared DMOAP films on titania surfaces as obtained by XPS and referencing the findings for the DMOAP-N/titania films obtained after 24 h immersion (labelled “DMOAP-N 24 h” here) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Table 9 – Contact angle results for DMOAP and ODPA films on smooth titania (freshly prepared . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Table 10 – Mean (SD) findings of E. coli growth attached to sample surface in 24 ha . 58 Table 11 – Changes of water contact angles obtained for thick and thin DMOAP films on titania substrates and for ODPA/titania after 24 h exposure to bacterial medium containing S. mutans and subsequent rinsing with alcohol . . . . 58 Table 12 – Normalized XPS signal intensity ratios for bisphosphonate samples ad- sorbed on TiO2, based on an octadecylphosphonic acid monolayer (ODPA SAM) previously published. . . . . . . . . . . . . . . . . . . . . . . . . . 69 Table 13 – Water contact angle (WCA) for the studied layers. . . . . . . . . . . . . . 70 Table 14 – BET measurements of prepared samples. . . . . . . . . . . . . . . . . . 80 Table 15 – Freundlich adsorption parameters obtained from experimental data. . . . 80 Table 16 – Mean surface roughness of prepared samples. . . . . . . . . . . . . . . . 83 Table 17 – Elements in atomic% measured by EDS. . . . . . . . . . . . . . . . . . . 83 Table 18 – Water contact angle (WCA) for pristine and samples prepared with 4 mM BP solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Table 19 – Surface elementary composition from XPS measurements. . . . . . . . . 85 Table 20 – FWHM for the contributions of XPS analyses. . . . . . . . . . . . . . . . 130 Table 21 – Water contact angle (WCA) for samples functionalized with 0.2 mM solutions132 List of abbreviations and acronyms pH potential of hydrogen TiO2 titanium dioxide HA hydroxyapatite BPs bisphosphonates BP bisphosphonate ATP adenosine triphosphate XPS X-ray photoelectron spectroscopy AFM atomic force microscopy ICP-MS inductively coupled plasma-mass spectroscopy DFT density functional theory QACs quaternary ammonium compounds DMOAP dimethyloctadecyl (3-trimethoxysilylpropyl) ammoniumchloride ODPA octadecylphosphonic acid AMC antimicrobial coatings SAMs self-assembled monolayers CAS chemical abstracts service sccm standard cubic centimeter per minute HPLC high performance liquid chromatography ETI etidronate ALE alendronate RIS risedronate Contents 1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2 GENERAL AND SPECIFIC GOALS . . . . . . . . . . . . . . . . . . . . 31 3 NEW DETAILS OF ASSEMBLING BIOACTIVE FILMS FROM DISPER- SIONS OF AMPHIPHILIC MOLECULES ON TITANIA SURFACES . . . 32 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3.2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3.2.2 Preparation of bulk DMOAP and ODPA for XPS investigation . . . . . . . 36 3.2.3 Surface preparation – film deposition . . . . . . . . . . . . . . . . . . . . 36 3.2.4 Solution preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.2.5 Layer formation on TiO2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.2.6 DMOAP adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.2.7 ODPA adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.2.8 Atomic force microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.2.9 Contact angle measurements . . . . . . . . . . . . . . . . . . . . . . . . 38 3.2.10 XPS characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3.2.11 Zeta potential measurements . . . . . . . . . . . . . . . . . . . . . . . . 40 3.2.12 Antibacterial assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.3.1 XPS characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.3.1.1 Bulk sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.3.1.2 Adsorbed layers on smooth titania substrates . . . . . . . . . . . . . . . . . 45 3.3.1.2.1 ODPA/titania films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 3.3.1.2.2 DMOAP/titania films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 3.3.1.2.3 Comparing ODPA/titania DMOAP/titania films . . . . . . . . . . . . . . . . . . . 51 3.3.2 Contact angle measurements . . . . . . . . . . . . . . . . . . . . . . . . 51 3.3.3 Zeta potential measurements . . . . . . . . . . . . . . . . . . . . . . . . 53 3.3.4 AFM investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 3.3.5 Antibacterial assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 3.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.5 Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 3.6 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4 BISPHOSPHONATES ON SMOOTH TIO2: MODELING AND CHARAC- TERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 4.2 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 63 4.2.1 Theoretical modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 4.2.2 XPS characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 4.2.3 Contact angle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 4.2.4 Zeta potential measurements . . . . . . . . . . . . . . . . . . . . . . . . 71 4.2.5 Atomic force microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 72 4.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 4.4 Supporting Information Summary . . . . . . . . . . . . . . . . . . . . 74 4.5 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 5 ADSORPTION AND X-RAY PHOTOELECTRON SPECTROSCOPY IN- VESTIGATION OF BISPHOSPHONATES ON TITANIA AND HYDROX- YAPATITE SURFACES . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 5.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . 76 5.2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 5.2.2 Synthesis of TiO2 solution, HA, and TiO2 particles . . . . . . . . . . . . . 77 5.2.3 Preparation of Ti substrates and film deposition . . . . . . . . . . . . . . 77 5.2.4 BP adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 5.2.5 Characterizations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 5.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 79 5.3.1 Effects of BP adsorption on TiO2 particles . . . . . . . . . . . . . . . . . 80 5.3.2 Structures and morphologies of prepared films . . . . . . . . . . . . . . 81 5.3.3 Effects of BPs adsorption on TiO2, HA, and TiO2+HA films . . . . . . . . 83 5.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 5.5 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 6 MICROBIOLOGICAL ACTIVITY OF BISPHOSPHONATES ON TITANIA, HYDROXYAPATITE AND COMPOSITE SURFACES . . . . . . . . . . . 90 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 6.2 Materials an methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 6.2.1 Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 6.2.2 Minimum inhibitory concentration (MIC) . . . . . . . . . . . . . . . . . . 91 6.2.3 Cell culture experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 6.2.4 MTT assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 6.2.5 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 6.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 93 6.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 7 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 APPENDIX 118 APPENDIX A – SUPPLEMENTARY INFORMATION CHAPTER 3 . . . 119 APPENDIX B – SUPPLEMENTARY INFORMATION CHAPTER 4 . . . 124 B.1 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 B.1.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 B.1.2 Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 B.1.3 Sputter deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 B.1.4 Layer preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 B.1.5 Atomic force microscopy measurements (AFM) . . . . . . . . . . . . . . 126 B.1.6 Contact angle measurements . . . . . . . . . . . . . . . . . . . . . . . . 126 B.1.7 XPS characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 B.1.8 Zeta potential measurements . . . . . . . . . . . . . . . . . . . . . . . . 127 B.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 B.3 X-ray photoelectron spectroscopy . . . . . . . . . . . . . . . . . . . . 130 APPENDIX C – SUPPLEMENTARY INFORMATION CHAPTER 5 . . . 131 1 Introduction Among the specialized connective tissues, bone tissue is the main component of the human skeleton. It protects the vital organs, produces blood cells, provides support to our body, and acts as levers for muscles. Additionally, bone tissue acts as a reservoir for ions such as calcium and phosphates, thus regulating the internal pH through homeostasis. Its structure is formed by an inorganic part, which mainly comprises phosphorus, calcium, and an organic matrix formed by collagen (mainly type I), proteins and proteoglycans (MAFFULLI; VIA, 2019). From a biological perspective, bone tissue is formed by three types of cells: os- teoblasts, osteocytes, and osteoclasts (MESCHER, 2018). Osteoblasts originate from mes- enchymal stem cells, and are responsible for producing organic matrix and depositing inorganic components for new bone formation. Osteocytes are former osteoblasts that help to control the levels of calcium and phosphorus ions; furthermore, they comprise 90% of the cells of a healthy skeleton. The main function of osteoclasts, multinucleated cells originating from macrophages, is bone resorption (MAFFULLI; VIA, 2019; MESCHER, 2018). These three cells are the main cells responsible for bone tissue physiology, which includes bone growth and resorption. This dynamic and continuous process of growth and resorption is also called bone turnover (PARFITT, 2002). In a healthy person, bone growth occurs through the secretion of collagen from osteoblasts, which is mainly located in the outward part of the bone, as shown in Figure 1. This secretion leads to the formation of a layer called osteoid. The mineral components are deposited through their interaction with glycoproteins and proteins such as osteocalcin, thus increasing the amount of Ca2+ under the surface. In addition, osteoblasts release phosphonate-rich vesicles, which work as a nucleus for crystal growth, resulting in the formation of hydroxyapatite crystals (Ca5(PO4)3OH) (MESCHER, 2018). This continuous secretion and crystallization leads to the formation of a new bone. Alongside the bone growth, bone resorption, which is regulated by osteoclasts, can be observed. First, these cells, which are in contact with the bone surface, corrugate their membrane. Then, a few enzymes are excreted along with acids, such as nitric and lactic acids. This process causes the dissolution of organic and inorganic matrix accompanied by the absorption of small particles by osteoclasts, lately being released into the blood (KHONSARY, 2017). Although the aforementioned process is in equilibrium, wherein no bone growth or resorption occurs, some situations, such as osteoporosis and traumas, alter this equilibrium. Generally, traumas can give rise to the need for a partial or total bone replacement. In this scenario, it is important to have an experienced doctor, a well-defined implant, and good 23 Figure 1 – Schematic representation of mineralization in bone matrix Source: Developed by Marina Cardoso based on (MESCHER, 2018) health. However, these conditions are not met always; thus, it is crucial to have an implant that can replace the bone and positively intervene in the healing process. Comorbidities, such as osteoporosis, diabetes, and Chron disease are related to implant failures (BLOCK et al., 2021; KASHI; SAHA, 2019). Additionally, smoking habits, alcoholism, and bruxism are associated with higher chances of implant failure (BLOCK et al., 2021; ANGELIS et al., 2017). For metallic implants, such as dental and hip implants, poor bone quality and overloading can be added as negative factors to the healing process. A review of the cases related to hip implant failure demonstrated the abovementioned for a short period (0-30 days), medium period (31 days to 6 months), and long period (6 months to 8.6 years) of implantation. The presence of comorbidities is listed as one of the factors that cause failure in all the scenarios (JOHNSEN et al., 2006). Although the effect of different types of commodities on the post-surgery is still being discussed. Evidently, the patient health and habits strongly influence the bone healing process after implantation (AGHALOO et al., 2019; BLOCK et al., 2021; ANGELIS et al., 2017). Thereby, the research and production of the multifunctional implant surfaces are crucial in reducing the implant failure rate. 24 Titanium is the frontrunner constituent element for metallic implants. Being one of the most abundant elements in the world, titanium is a transition metal that can be applied aeronautically to the biomedical equipment. Its use as a constituent for implants starts with the pioneering work of Brånemark and coworkers in the late 1960s (ADELL et al., 1981). Titanium and its alloys presented properties such as toughness, corrosion and wear resistance, and biocompatibility (KAUR; SINGH, 2019; SIDAMBE, 2014). A detailed discussion related to the mechanical, structural, and machine processes of the titanium alloys can be found in the selected reviews (KAUR; SINGH, 2019; LI et al., 2020; NICHOLSON, 2020; SIDAMBE, 2014). Excluding the mechanical properties that must be attended for metallic implants, biocompatibility and other biological aspects are surface-dependent. In other words, properties such as the chemical composition and roughness of the implant surface directly affect the cell response (RATNER; CASTNER, 2020). Titanium is a reactive metal, thus when it is exposed to air, an oxide layer, also known as the passivation layer, is created. Such spontaneous film has a thickness of approximately 4-10 nm. Although its presence increases the corrosion resistance, researchers have been working on different strategies for the growth of this oxide layer, including plasma deposition, sol-gel, and chemical etching (CHOUIRFA et al., 2019; NICHOLSON, 2020). The titanium dioxide layer commonly presents several hydroxyls on its surface, usually, at a concentration of approximately 4.9–12.5 nm−2 (HANAWA, 2019). These groups may be increased by chemical or physical processes. In all the cases, the presence of OH species leads to good biocompatibility of the titanium-based materials (HANAWA, 2019). Once in contact with the biological medium, hydroxyl groups expose Ti+ or create Ti-O− species while leaving the surface. The increase of Ti-O− may favor the adsorption of proteins, such as albumin, integrin, and cytokine. After protein adsorption, neutrophils, monocytes, and macrophages adhere to the surface and start the recognition process. Two different responses can be triggered depending on the environmental conditions . In the first case, the foreign body reaction keeps evolving, leading to the encapsulation of the implant by a fibrous capsule, because the macrophages were not able to phagocyte the whole surface. In the second scenario, the collagen fibers are deposited; however, the signalization process is different. In addition to the macrophages’ action, osteoblasts start to act using the fibers as a scaffold for new bone formation (BRUNETTE et al., 2001; HANAWA, 2019; JOHN et al., 2016; YOSHINARI et al., 2002). Among the techniques for titanium dioxide deposition, the sol-gel route is the most suits one for this purpose. Compared to other methodologies, the sol-gel has advantages such as low cost compared with sputtering, low-temperature processing, chemical control, and precise microstructure. Additionally, due to the high surface area, materials prepared using this methodology present higher levels of biocompatibility (OWENS et al., 2016). One of the methodologies for the synthesis of TiO2 by sol-gel uses titanium isopropoxide as a precursor (TRINO et al., 2018b), the synthesis mechanism is displayed in Figure 2. This 25 alkoxide is hydrolyzed in an acidified aqueous medium, resulting in a condensation reaction, which transforms Ti(OH)4 in Ti(OH)4O+H2 through solvation. Polymer chains of Ti-O-Ti are formed through the olation process, owing to the presence of Ti-OH+-Ti species. The olation process occurs due to nucleophilic substitutions, in which Ti4+ is the nucleophile. The presence of water and alcohol in the medium allows the removal of the H2O+ and R-OH species. Peptization is the subsequent step when the aggregates start to break, forming a sol of small particles. Finally, solvent evaporation leads to the formation of the gel. The particle size of the forming gel can be controlled using a surfactant. Regarding its structure, titanuim dioxide exist mainly in three different structures: brookite, rutile and anatase. Rutile is the thermodynamically most stable structure and commonly employed for biomedical applications (DIEBOLD, 2003; TRINO et al., 2018a). Anatase has a higher photoactivity compared with rutile and brookite has a stability between rutile and anatase (LUTTRELL et al., 2014; ZHANG; BANFIELD, 2014). Here, rutile was choosen as main phase for TiO2. Figure 2 – Synthesis mechanism of titanium dioxide by sol-gel route using titanium iso- propoxide as precursor. Source: Developed by the author Since hydroxyapatite is one of the bone constituents, its presence combined with the hydroxyls from titania can also improve the biological performance of the deposited films, compared to the activity of the individual films (HE et al., 2008; WANG et al., 2018). Thus, using hydroxyapatite (HA) films with titanium dioxide, is a suitable strategy for coating metallic implants; however, the adhesion of HA over the titanium surface is poor, which may lead to detaching or cracking under certain levels of loading (HAN; YU; ZHOU, 2008; HARUN et al., 2018). A convenient strategy to overcome this characteristic is the deposition of the mixed films of TiO2 and hydroxyapatite. Previous studies have shown that, compared to the pure HA coating, composite films of TiO2/HA may present a 40% higher adhesion force (HAN; YU; ZHOU, 2008). The composite coatings can be produced by adding the 26 hydroxyapatite powder to the TiO2 gel; simultaneous synthesis; and a multilayer approach, in which the HA is deposited over the titanium dioxide layer (MILELLA et al., 2001; RAMIRES et al., 2001; SIDANE et al., 2017). After the deposition of the oxide or composite film, a further step may be performed on the surface, which includes the addition of an organic layer on the surface. This adsorption process has demonstrated the ability to increase the biocompatibility on surfaces, in addition to improving other properties such as hydrophilicity, corrosion, and tribocorrosion resistance (BRONZE-UHLE et al., 2019b; TRINO et al., 2018b; TRINO et al., 2018a). This layer can be a compound with specific properties and characteristics such as bifunctional molecules, proteins, peptides, drugs, and even polymers. Moreover, bisphosphonates (BPs) are a class of molecules suitable for layer deposition. Similar to pyrophosphate, bisphosphonates are a class of molecules used for the treatment of osseous diseases, such as osteoporosis and Paget’s disease. Instead of central oxygen, BPs have a P-C-P structure that presents two substituents, R1 and R2, as shown in Figure 3. The presence of a central carbon provides higher stability and increases the resistance to hydrolysis compared to its inorganic analogous (ROGERS; MöNKKöNEN; MUNOZ, 2020). Generally, the R1 chain is a hydroxyl group and the R2 may vary from a methyl group to heterocyclic rings (Figure 3 b,c,d). In physiological conditions, bisphosphonates exist mainly in their ionic state, which can vary according to the present chains (BEDOYA et al., 2017; LIN, 2019; ROGERS; MöNKKöNEN; MUNOZ, 2020). Figure 3 – a) General structure of bisphosphonates, b) etidronate, c) alendronate and d) risedronate linear structure. Source: Developed by the author As mentioned previously, bisphosphonates are usually employed to regulate the bone metabolism, which can be compromised for different reasons. Due to its ionization, bisphosphonates act through the chelation of Ca2+ of the body, which leads these molecules to rapidly vanish from circulation due to their strong affinity towards the hydroxyapatite crystals (RUSSELL et al., 2008). Once these molecules are immobilized on the HA surface, 27 they have different mechanisms of action that are not fully understood. To simplify, the main mechanisms are as follows: (1) when osteoclast lays on the surface and starts the secretion of acids, BPs are released along with calcium crystals; (2) the material is internalized and bisphosphonates are used to synthesize adenosine triphosphate (ATP) owing to the presence of phosphonates groups. However, due to its different structure, the produced ATP analogue is toxic, leading to the death of osteoclast; (3) the presence of BPs in the prenylation of proteins is important for osteoclast function, which also induces cell death. The process is summarized in Figure 4 (CUI et al., 2019; RUSSELL et al., 2008). The knowledge on these mechanisms is still unclear; however, studies showed beneficial effects of BPs on the osteoblast cultures (DOLCI et al., 2019; MALAVASI et al., 2016). Conversely, excessive or long-term use of bisphosphonates, which are administered orally or intravenously, is associated with jaw osteonecrosis, an adverse effect which is not fully clarified (ABTAHI et al., 2013). Considering the patients with implants, a literature review demonstrated that there is no evidence of the safe use of bisphosphonates before or after the implant surgery (GUAZZO et al., 2017). In other words, bisphosphonates cannot be administrated orally or intravenously as a procedure to reduce the implant failure. The local use of bisphosphonates is an emerging alternative that aims at avoiding undesirable effects and maintaining its osteogenic activity (ABTAHI et al., 2013; KHAMIS; ELSHARKAWY, 2018). Prior to its usage, the drug can be locally applied through injection or deposited on the implant surface. The immobilization of bisphosphonates on the implant surface can be achieved by immersing the implant in a solution containing the drug. This is a simple procedure compared to administering the injections, which may require health professionals to perform the method. As mentioned above, the BPs have a strong affinity for HA, which may justify most investigations toward the HA/BPs systems. Various researchers and groups have demon- strated the adsorption mechanism of the bisphosphonates on hydroxyapatite, and the stability of this system under physiological conditions (BOSCO et al., 2015; FORTE et al., 2017; FORTE et al., 2019; NANCOLLAS et al., 2006). These investigations highlight the impor- tance of the HA/BPs systems. However, to effectively use these drugs as implant modifiers, the mechanism of adsorption, desorption, stability, and other aspects of oxide/BPs systems must be scrutinized. Mainly TiO2/BPs helps to produce implants containing bisphosphonates under the surface. Few studies have shown the characteristics of titania/bisphosphonates arrangements from the physicochemical perspective. To achieve this goal, analytical tech- niques, such as X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), zeta potential, contact angle, and other surface-sensitive techniques are necessary. (ROJO et al., 2016) immobilized alendronate on Ti6Al4V. The contact angle results showed that the presence of the adsorbent reduces the contact angle for water and increases the values of surface free energy. The XPS results indicated that alendronate binds on the 28 Figure 4 – Effects of BPs on bone metabolism. A) Transformation of non-N-BPs into cytotoxic ATP analogs prompts osteoclast apoptosis, while N-BPs induce osteoclast apop- tosis by inhibiting FPPS. B) BPs modulate the expression of osteoclast-related genes and promote the expression of osteogenicrelated genes. C) BPs restrain BMSC adipogenic differentiation and promote their osteogenic differentiation. D) BPs inhibit apoptosis of osteoblasts and osteocytes. Source: (CUI et al., 2019). Published under the license number 5378781404773. surface through phosphonates and nitrogen. However, the authors did not provide a strong evidence of the binding mode through XPS, mainly using O1s and C1s high-resolution spectra. Additionally, as indicated by the authors, there is no sufficient proof for monolayer formation; for instance, the lack of layer thickness. Considering that the size of alendronate is approximately 0.94 nm (KARLSSON; ATEFYEKTA; ANDERSSON, 2015), it is reasonable to assume that the presence of a monolayer would be insufficient to cause the biological response presented in the referred study. In this case, the molecules could rapidly vanish and/or be absorbed by the cells. thus, the immobilization efficacy lasts no longer than a few hours. 29 A different study from (ZHENG; NEOH; KANG, 2017) used alendronate immobilized on the Ti surface. The authors treated the surface with two different methods: plasma oxygen, and silanization. This study presents the survey spectra of XPS, which demonstrated the presence of nitrogen and phosphorus, the characteristics of alendronate composition. How- ever, the high-resolution spectra were not presented, precluding the statements concerning the binding mode. Furthermore, studies demonstrated that the presence of alendronate reduce s the water contact angle of the surface. The immobilization of alendronate does not increase the surface nano roughness, as demonstrated by atomic force microscopy. Using inductively coupled plasma-mass spectroscopy (ICP-MS), the authors showed that the amount of adsorbent on the surface may vary from 308 to 1655 ng/cm2, thus, indicating a layer thickness of 5.7-32.5 nm, which is different from the expected value of 0.94 nm for a monolayer. Additionally, depending on the immobilization procedure, the alendronate layer can release a maximum of 400 ng/cm2 in 14 days of incubation in water. This result suggests the presence of a synergetic effect between the adsorbed and released molecules, as the same work mentioned that a modified titanium surface has a better biological performance in terms of cell survival and cell metabolism. Using the density functional theory (DFT) (PETROVIć et al., 2020) discussed that alendronate is adsorbed on the TiO2 surface through a competition between -NH2 and -PO3H groups, leading to two possible conformations. In the first one, the alendronate binds through amino and phosphonate; in the second one, it is through phosphonate only. Although this discussion can elucidate the aspects of the adsorption process, the simulation does not include the protonation states of the molecule. As indicated by the different studies, bisphosphonates exist mainly in their ionic state at physiological pH (BEDOYA et al., 2017; FERNáNDEZ; ORTEGA-CASTRO; FRAU, 2017; KE et al., 2016). The authors associated free nitrogen and phosphonates with the decrease of water contact angle. Electrochemical measurements indicated that the alendronate layer increases the corrosion resistance and may remain stable for seven days. A study by (BRONZE-UHLE et al., 2019a) showed the influence of UV irradiation on the bisphosphonates adsorption. In this work, the TiO2 surface was irradiated with UV light before immersion in the bisphosphonate solution. The XPS results indicate modifications in the alendronate and risedronate adsorption. Without the UV irradiation, the BPs can bind through the nitrogen-containing and phosphonates groups. While on an irradiated surface, the interaction surface-BPs occur through phosphonate groups alon e. In agreement with other studies, a decrease in water contact angle was observed. The effects of UV radiation before the bisphosphonates adsorption on titania is not fully recognized. (BRONZE-UHLE et al., 2019a; KIM et al., 2017) demonstrated that this process modifies the binding mode and may improve the biological performance. (KIM et al., 2017) showed that the implants irradiated with UV light before immersion in alendronate 30 solution presented better biological results compared to the non-irradiated titania, including cell viability, adhesion, and alkaline phosphatase. Different authors studied the adsorbed bisphosphonates on surfaces that are not hydroxyapatite, although most of them focused on the biological performance of such modification. In this aspect, they reported that the immobilization of BPs can improve the biological markers and increase cell proliferation, survival, and adhesion (CHA; LEE; LEE, 2018; MALAVASI et al., 2016; YOSHINARI et al., 2001). This important information may contribute to the development feasible implants covered with BPs. Nonetheless, some topics still remain unclear. Since such results are surface- dependent, well-characterized surfaces are crucial. As discussed by different authors, a well-performed surface characterization helps to understand how the adsorbed molecules can increase the surface biocompatibility. Therefore, oxide/BPs and composite/BPs systems are good candidates for extensive characterization. 2 GENERAL AND SPECIFIC GOALS Considering the above mentioned discussion, the present work has as general goal to elucidate aspects of bisphosphonates adsorption on TiO2, HA and HA+TiO2 surfaces. From bisphosphonates, etidronate, alendronate and risedronate were chosen as surface modifiers, as shown in Figure 3 b,c,d. As hypothesis, this works try to verify if bisphosphonates can positively affect cell growth on the proposed surfaces. As specific goals, we propose preparation of TiO2, hydroxyapatite and composite surfaces, togheter with an extensive characterization of bisphosphonates layers using mainly X-ray photoelectron spectroscopy. Additionally, we propose to obtain a adsorption curve of bisphosphonates on titania (anatase and rutile). 3 New details of assembling bioactive films from dispersions of amphiphilic molecules on titania surfaces This chapter was originally published on RSC Advances, 2020, 10, 39854-39869 as original article, the manuscript is available through the DOI: 10.1039/D0RA06511K. 3.1 Introduction The design of novel functional biomaterials depends on tailoring their multiscale structures (WEI et al., 2014; STAPELFELDT et al., 2019). Surface modification is a suitable strategy to enhance the properties of biomaterials such as biocompatibility and antibacterial efficacy (CORRALES-UREñA et al., 2020). Different procedures are well established, such as depositing silver as an antibacterial component or immobilizing functional molecules on medical products (BORCHERDING et al., 2019; BRONZE-UHLE et al., 2019a).Aiming to introduce antibacterial properties to medical devices, a valid alternative is grafting chem- ically reactive quaternary ammonium compounds (QACs), such as dimethyloctadecyl (3- trimethoxysilylpropyl) ammonium chloride (DMOAP). DMOAP has a quaternary ammonium group that is separated by a propylene spacer from a hydrophilic tri-methoxy silane group and bound to an aliphatic octadecyl chain (C26H58NO3SiCl), which is responsible for its hydropho- bicity (MENG et al., 2015).ODPA (octadecylphosphonic acid) also contains a hydrophobic chain, and its hydrophilicity is due to a protonated or deprotonated phosphonate group. The immobilization of ODPA can also form antibacterial response on surfaces (ZHANG et al., 2017). Structural formulas of linear DMOAP and ODPA conformations are presented in Fig. 5. Hydrophobic surfaces can be suitable for biomedical devices to avoid protein adsorption, increase the surface charge in contact with aqueous fluids, and establish an antimicrobial ef- fect (ZHANG et al., 2017; ZHANG; WANG; LEVäNEN, 2013; BRAUNWARTH; BRILL, 2014; GRACE et al., 2016). Modifications of surface charge play an important role in achieving antimicrobial efficacy in different bacteria-containing media (KAUR; LIU, 2016). Although a long alkyl chain could increase the surface hydrophobicity and influence bacteria adsorption, the presence of a cation induces electrostatic interaction with the bacteria phospholipid bilayer (DOGRA et al., 2012). As reviewed by Elena and Miri (ELENA; MIRI, 2018), QACs can be immobilized on different surfaces, from cotton fibers to metal oxides, apart from being incorporated in the synthesis of resins for dental applications (ANTONUCCI et al., 2012). Quaternary ammonium https://doi.org/10.1039/D0RA06511K 33 Figure 5 – Linear structure of (a) DMOAP and (b) ODPA. Source: Developed by the author compounds present antibacterial activity even when their head groups are hydrolysed in a solution; however, non-immobilized QACs are often active surface water contaminants and can potentially cause environment damage (BOETHLING, 1984). Thereby, immobilization of QACs on a surface while largely maintaining their activity is a desirable but challenging procedure, which aims to provide a non-release system and avoid environment impact (NEU, 1996; OOSTERHOF et al., 2006). For instance, Meng et al. demonstrated that when immobilized on cellular membranes, DMOAP can maintain its antibacterial efficacy in various conditions, such as pH of 3.5 and temperature of 50°C (MENG et al., 2015). Despite its attested antibacterial efficacy, the mechanism of action of DMOAP has not been fully elucidated. The mechanism has been described in the following manner: attraction between the positively charged DMOAP layer and the negatively charged bacteria cell membrane occurs supported by van der Waals interactions between the respective DMOAP or phospholipid hydrophobic tails (ASRI et al., 2014). The electrostatic attraction leads to an ionic interaction causing the transport of divalent cations (Ca2+ and Mg2+), inducing membrane disruption and consequent death of the bacteria (ASRI et al., 2014; BELKHIR et al., 2017). The surface charge density induced by the ammonium cation plays a crucial role in the antibacterial activity. Asri et al., using AFM measurements, reported that immobilized QAC has a strong interaction with Staphylococcus epidermidis, and the force of interaction with the bacteria surface was five to six times greater than usually found for bacteria adhesion on surfaces, which are in a range of 1 nN (ASRI et al., 2014). He et al. observed that when E. coli attaches on a DMOAP surface, a distortion of the cell membrane leads to its rupture (HE et al., 2016). 34 On the other hand, immobilized ODPA provides antibacterial properties due to the increased surface hydrophobicity, consequently avoiding the adsorption of some bacteria (ZHANG et al., 2017). Zhang et al. reported that ODPA immobilization on titania nanotubes not only avoids bacterial adherence but can also modulate the release of Sr and Zn when these are dopants of the nanotubes (ZHANG et al., 2017). ODPA is irreversibly adsorbed on Ti0.5Al0.5N surfaces forming a non-release system, as reported by Theile-Rasche et al (THEILE-RASCHE et al., 2020). In addition to characterization, useful tools for revealing the arrangement of DMOAP and ODPA layers on surfaces are contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. DMOAP and ODPA modify the wettability of the surface due to their long alkyl chains. Besides the binding situation and area density of the functional groups of those molecules, quaternary ammonium for DMOAP and phosphonate for ODPA can be detected by XPS (ASRI et al., 2014; ZHANG et al., 2017; THEILE-RASCHE et al., 2020; DAI et al., 2014). For tailoring the physicochemical properties in aqueous environment, such as zeta potential and wettability of the layer systems formed by immobilized DMOAP and ODPA, details about the adsorption process are important to understand. To elucidate the effects of process parameters in forming bioactive layers, combining microscopic and spectroscopic studies with nanoscale sensitivity and a layer build-up on flat and chemically well-defined substrates is innovative, as was shown for designing highly active antifouling layers (KULKA et al., 2019) and antimicrobial films, promoting protein adsorption (FABRE et al., 2018), and verifying the influence of electrolytes on contact angle (AL-ZAIDI; FAN, 2018). For achieving antibacterial contact-activity, numerous approaches for tailoring sub- strate surface properties have been reported. Surface structure, wettability, composition, and time-dependent release properties in aqueous environment have been generally assessed. These studies revealed that increased antimicrobial activity is promoted by adjusting the surface roughness instead of providing smooth substrate surfaces; introducing amphiphilic moieties in films that will be in contact with phospholipid-based bacterial membranes; inte- grating molecular groups that affect the surface charge like QACs; or facilitating the release of metal ions from bulk film regions that are not exposed to the convective transport for permitting longer-term activity of antimicrobial coatings (AMC). The underlying antimicrobial strategies may comprise several components. Among them, providing anti-adhesive surfaces to avoid or retard microbial attachment (AGUIAR et al., 2019), contact-active surfaces that are based on immobilized and not released active species (CORRALES-UREñA et al., 2020; UREñA et al., 2015), and biocide-releasing surfaces (ADLHART et al., 2018). Tailoring strongly attaching films composed of amphiphilic molecules with respect to their antibacterial activity has been reported based on approaches considering thick films (HSU et al., 2020; FORMAN et al., 2016) and polymeric (HE et al., 2016) or hybrid 35 (ASRI et al., 2014) bulk materials containing quaternary ammonium species. For such substrates, a substantial conformational freedom of the molecular entities exposing the quaternary ammonium species may be expected in a swollen state in the presence of aqueous environments containing bacteria (HE et al., 2016). In addition to chemical modifications, the topography of a surface can significantly affect its hygienic status, either beneficially (reducing microbial retention) or negatively (in- creasing retention) (ADLHART et al., 2018). Findings of surface charge densities around 1.5 x 1015 cm−2 or even 1016 cm−2 for monovalent ammonium cations are based on calculations considering the geometric surface area of rough substrates rather than on their active surface area, since monolayer densities do not exceed 5 x 1014 atoms cm−2, even of unbranched nonanethiols on gold (CAMPOSEO et al., 2006; JIAO et al., 2017). The present study aims to achieve a more profound understanding about material- related properties governing a contact-killing ability from the interaction between bacteria and substrates containing quaternary ammonium species by assessing dynamic effects in the bacteria/QAC interface. Properties of silane-based layers and their mechanism of immobilization need to be explored to devise deposition processes and exploit their versatility for surface modification (PLETINCX et al., 2019). In addition, systematic studies for governing the contact formation and its effect by tailoring the substrate surface were performed. A versatile and well-controlled multi-step approach was developed for designing the AMC surface. Starting from a topologically well-defined substrate that may be smooth or micro- structured, thin biocompatible and non-cytotoxic titania nano-films were deposited as a base for the application of AMCs. As immobilizing QACs in thin layers may strongly restrict conformational freedom of the molecules, DMOAP films with different thickness were studied starting from the monolayer regime to demonstrate the versatility of our approach. Studies relating layer structure and its biological activity are scarce and need to be clarified (LIU et al., 2020); thus, this work explores this gap in the literature, showing well characterized layers before and after biological assays. Smooth substrates were used to restrict the potential bacteria/QAC interface to the geometrical surface area. Inorganic oxidic titania substrate material was also used to exclude swelling effects upon exposure to aqueous environments. Finally, ODPA SAMs were used, which like DMOAP contain hydrophobic octadecyl molecular tails. The orientation of amphiphilic molecules in such well-ordered monolayers provides a reference for working out the effects of molecular orientation observed in thin DMOAP films. 36 3.2 Materials and methods 3.2.1 Materials Silicon wafers (diameter of 100 mm) from Si-Mat and glass slides (75 x 25 mm) from Thermo Scientific® were used as substrates. Titanium dioxide was obtained from a metallic titanium target that was used for sputter-deposition in an oxygen containing atmosphere. A methanol-based mixture of 42 wt% dimethyloctadecyl(3-(trimethoxysilyl)propyl)ammonium chloride (DMOAP) (CAS 27668-52-6) and 8 wt% of (3- chloropropyl)trimethoxysilane from SIGMA-ALDRICH® and octadecylphosphonic acid (ODPA) (CAS 4724-47-4) from TCI® Germany were employed for the layer formation experiments on sputter-deposited TiO2 on silicon wafers and glass. Ethanol 99% (CAS 64-17-5) from Labochem® was used as solvent to prepare DMOAP and ODPA solutions. 3.2.2 Preparation of bulk DMOAP and ODPA for XPS investigation To prepare samples for XPS investigations and bioactivity tests, DMOAP was dropped as received in a silicon wafer, and after evaporation of the solvent the obtained specimen was henceforth called “bulk DMOAP” sample. A second sample was prepared by exposing the obtained solid deposit product to water at room temperature for distinct periods and subsequently dried. The thus prepared samples were called “water exposed DMOAP”. As a reference for the biological assay, DMOAP was dropped on titania surface and the solvent was left to dry in air. The procedure was repeated twice. The final sample named “Blot DMOAP” was a thick layer heated overnight at 100°C in an oven at ambient atmosphere. For preparing the so-called “bulk ODPA” samples, a small quantity of the ODPA product powder was dissolved in 99% ethanol to obtain a turbid suspension. A drop was placed on a silicon wafer and gently blown dry using an air stream generated by a hand bellow to remove the solvent excess. 3.2.3 Surface preparation – film deposition A silicon wafer was cut in two different sizes, 3 x 5 mm and 20 x 20 mm, and the respective pieces were used for XPS measurements and biological assays, respectively. After the cutting procedure, the substrates were ultrasonically cleaned in water for 10 minutes. Glass slides were used as received. Subsequently, the substrates were placed in a vacuum chamber then pumped until reaching a pressure of 10−5 hPa. The argon flux was adjusted to 120 sccm. Finally, the titanium dioxide deposition started. First in the deposition process, the target was sputtered without the presence of oxygen for 30 s. Afterwards, oxygen was slowly added into the chamber, using a flux of 9 sccm, for 900 s. A power of 2300 W and maximum bias of 765 V were applied. For all adsorbents, the layer formation experiments 37 were performed five minutes after sample removal from the chamber. 3.2.4 Solution preparation Two different solutions containing DMOAP were prepared, similar as described by Torkelson et al (TORKELSON et al., 2012). A volume of 0.6 mL of 42 wt% DMOAP solution in methanol was diluted in 49.4 mL of 99% ethanol to produce the first working solution. For the second solution, the same procedure was followed and 10 drops (approx. 50 µL) of 1 M aqueous hydrochloric acid (previously prepared) were added after mixing the alcoholic liquids. The solutions were called “DMOAP-N” (without acid addition) and “DMOAP-A” (with acid addition). ODPA solution was prepared as follows: 167.25 mg of ODPA were dissolved in 50 mL of 99% ethanol, establishing a concentration of 1 mM. All immersion experiments of titania-coated substrates were performed approximately 30 minutes afer solution preparation. In this freshly prepared state, the solutions visually appeared transparent, clear, and colourless and remained as such during the immersion procedures. 3.2.5 Layer formation on TiO2 The procedures described below were used for all titania-coated substrates. All samples were gently rinsed (either in distilled water or ethanol) after immersion in DMOAP and ODPA solutions, and identified as “water rinsed” or “ethanol rinsed”, respectively. 3.2.6 DMOAP adsorption To attach DMOAP on the titanium dioxide surfaces, the substrates were immersed for 10 minutes or 24 hours at room temperature for each DMOAP solution. After the submersion time, the samples were removed from the solution and gently rinsed using distilled water from a wash bottle. Two distinct drying procedures were employed. One method consisted in blowing the sample surface using clean compressed air after the washing procedure. For the second method, the samples were subsequently placed in an oven and completed drying in air at 100 °C overnight. To clarify the achieved samples, the descriptions of the preparation label and conditions are summarized in Table 1. 3.2.7 ODPA adsorption ODPA was adsorbed also through sample immersion in ODPA solution for 18 hours. Additionally, the samples were removed and either of the two above-described washing 38 Table 1 – Parameters of immersion, drying method, and sample name for DMOAP/titania films prepared from two different DMOAP solutions: DMOAP-N (without acid addition) and DMOAP-A (with acid addition) Solution Immersion time Drying method Sample name DMOAP-N 10 minutes Blown using compressed air TiO2/DMOAP-N 10 min Heated in an oven at 100 °C in air overnight TiO2/DMOAP-N 10 min heated 24 hours Blown using compressed air TiO2/DMOAP-N 24h Heated in an oven at 100 °C in air overnight TiO2/DMOAP-N 24h heated DMOAP-A 10 minutes Blown using compressed air TiO2/DMOAP-A 10 min Heated in an oven at 100 °C in air overnight TiO2/DMOAP-A 10 min heated 24 hours Blown using compressed air TiO2/DMOAP-A 24h Heated in an oven at 100 °C in air overnight TiO2/DMOAP-A 24h heated Source: Developed by the author procedures was followed. All samples were blown dry using air from a hand bellow to remove the excess solvent. 3.2.8 Atomic force microscopy Nanomechanical surface properties were assessed applying a Bruker Dimension Icon3 with a Nanoscope VSPM control unit and the software NanoScope V9.40R1. The cantilever and tip used were selected according to the surface properties (XSC11- Pt, MikroMesh, force constant 40 nN nm−1, tip radius < 20 nm). The calibration of the force constant was done by analyzing the thermal noise in combination with the Sader method (SADER et al., 2016).The sensitivity was calibrated with the help of an Al2O3 substrate. The tip radius was determined using the image of a nano rough Ti reference sample. The quantification was carried out according to a modified Villarubia method that was implemented in NanoScope (VILLARRUBIA, 1994). The QNM mode was used to quantify the nano-mechanical properties. For this purpose, the cantilever was excited non-resonantly at 2 kHz with amplitude of approximately 15 nm and indented into the surface with a depth up to a maximum of 2 nm; therefore, the indentation depth was approximately 10 nm. Height profiles were obtained using a Nanosurf easyscan AFM device. The images were taken in a resolution of 512 x 512 pixels in different sizes. For this, the non-contact mode (variable force) was used with a scan line ratio of 2 seconds for each image and a cantilever with a force constant of 40 nN nm−1. The post treatment was performed using the Gwyddion 2.55 software. First, a step line correction was applied. Then, the rows were aligned using a polynomial degree equal to five, and finally the strokes were corrected. The tip was a Tap190Al-G from Budget Sensors with a resonant frequency equals to 190 kHz. 3.2.9 Contact angle measurements Contact angle measurements were performed immediately after the drying proce- dures. The analysed samples were TiO2 sputter- deposited on glass slides. The measure- 39 ments were evaluated using the mobile surface analyser – MSA from KRÜSS – at room temperature. HPLC quality water and diiodomethane were used as probe liquids, and 2 µL of each liquid were simultaneously and automatically dropped on the surface. Then, the contact angle for each drop was recorded using the tangent method. Two distinct regions of the sample were analysed, and the mean contact angle for each liquid was calculated. Changes of apparent water contact angles as a consequence of exposing DMOAP coated titania specimens to aqueous bacteria medium were recorded using a goniometer based (OCA15 Plus, Data Physics Instruments, Germany) approach and applying the sessile drop technique. For each measure- ment, 5 µL drops were formed using HPLC-grade water (Acros Organics, Germany) and the subsequent contact angles were taken and analysed by the software SCA202 (Data Physics Instruments, Germany). The reported contact angles are an average of at least three measurements for each sample. 3.2.10 XPS characterization XPS characterization was carried out using a Kratos AXIS Ultra system with a monochromatized Al Kα X-ray source (energy hν = 1486.6 eV). The base pressure of the analysis chamber was approximately 6 x 10−8 Pa. Spectra were acquired in the constant analyser energy mode using pass energies of 160 eV and an energy step of 0.444 eV for survey spectra and 20 eV and energy step of 0.066 eV for detail scans. Spectra fitting was performed using CasaXPS (v2.3.18, Casa Software Ltd). First, the binding energy scale of the spectra was aligned using the C1s signal at 285.0 eV of the aliphatic hydrocarbon species. Then, the signal area to analyse was set using a Shirley-type or linear background. Peak components were adjusted using the line shape GL(30). The full widths at half maximum (FWHM) for the C1s components were set to be equal. The created areas were fitted using the Marquardt–Levenberg algorithm. Based on the signal attenuation, the thickness of the adsorbed layers was estimated using the equation d = ln(y).l, where d is the thickness of the adsorbed layer, y is the ratio between the area of the Ti2p peak from the coated sample and the pristine sample, and l is the inelastic mean free path of the electrons in the organic film (2.8 nm for Ti2p). For such estimation, the surface coating was assumed to be homogeneous (WEI et al., 2014). A thickness of 0.6 nm was considered for the adventitious carbon layer on the titanium substrates (MANGOLINI et al., 2014). An estimated layer thickness of an adsorbate was calculated based on the assumption that the adventitious carbon layer was replaced by the adsorbate layer. Atomic surface concentration values were calculated based on the simplifying geometric model assumption that the sample surface is homogeneously composed. 40 3.2.11 Zeta potential measurements Zeta potential measurements were performed with different titanium dioxide samples deposited on glass slides. The values were recorded using the SurPASS™ 3 device from Anton Paar at room temperature. The adjustable gap cell was used, and the gap adjusted to 100 mm. The pH was adjusted using 0.05 M solution of potassium hydroxide and 1 M solution of hydrochloric acid, respectively. The pH step was set to 0.75 in range from pH 8 to pH 4 (in this order) and measured with a pH meter sensor innate to the equipment. Before the measurements, the samples were stored for 24 hours in 2 hPa at room temperature. The error bars were estimated based on three distinct 3.2.12 Antibacterial assays Escherichia coli bacterium (strain DSM 10290) was cultured in LB broth medium at 37°C in aerobic conditions for 24 h. The growth culture suspension was centrifuged twice at 13000 rpm for 5 minutes and suspended in PBS buffer. The optical density (OD) was measured at 600 nm in UV-Vis Specord 200 Analytic Jena spectrophotometer and the concentration was adjusted to 1 x 107 CFU m−1 (with 0.06 OD corresponding to 2 x 108 CFU mL−1) in minimal medium (1 : 100 of LB broth medium in PBS buffer). In a UV2 Sterilizing PCR Workstation, at room temperature, the samples (n = 3) were placed in sterile Petri dishes and received sprays of isopropanol 70% . The Petri dishes lids were opened, the working bench was closed, and the UV light was turned on for 30 minutes to sterilize the samples. Afterwards, 50 mL of bacterial solution were applied over each sample surface and a sterilized cover glass slip (18 x 18 mm) was placed to spread the drop over the substrate, according to the ISO 22196. Moreover, 2 mL of PBS was pipetted in the outlines of the Petri dishes to prevent evaporation. After incubation for 4 h at 37°C, the cover glass slips were removed, and samples were carefully washed twice with distilled water to remove non-adherent or weakly adherent bacteria. The rinsed water was kept in a 2 mL centrifuge tube for further analysis. The samples were placed into a 50 mL centrifuge tube, which was filled with 15 mL of PBS buffer. The tubes were sonicated (Ultrasonic bath Sonorex RK100 Bandelin Electronic, Berlin, Germany) for three minutes and vortexed (Minishaker MS2, IKA – Werke GmbH & Co. KG, Staufen, Germany) for one minute to allow detachment of the adherent bacteria. The 24 h kinetic of both the non-adherent bacteria (from the rinsed water) and the bacteria detached from the sample were evaluated using Mith- ras LB 940 Microplate Reader (Berthold Technologies Ltd). Accordingly, 150 µL of each bacterial solution + 50 µL LB broth medium were pipetted in a well of a 96-well cell culture plate, which was placed in the device with all parameters set in the same conditions of the bacterium culture. The bacterial growth was related to the bacteria adhered on the surface of the samples, which after ultrasound and vortex remained alive and healthy to continue growing in suitable nutrients and temperature conditions. The bacterial growth from the rinsed water indicated a 41 bacteriostatic effect of the sample, which inhibits the initial bacteria adhesion and further biofilm formation. Testing with Gram-negative E. coli was introduced to the experimental design be- cause DMOAP immobilized on polymers exhibited comparatively low minimum inhibitory concentra- tions (MIC) against E. coli and Gram-positive S. aureus as compared to C. albicans fungus (LI et al., 2016). Gram-negative S. mutans was chosen to be part of the conceptual design including bulk DMOAP specimens because of its relevance for dental research and considering the paramount objective was to identify if DMOAP shows an antibacterial effect against S. mutans. 3.3 Results and discussion From the XPS spectroscopic point of view, the approach presented in this study profits from opting for smooth titania films. Moreover, on smooth substrates, the morphology of organic adsorbates can be accessed (WEI et al., 2014). In comparison to silica or alumina adsorbents exhibiting contributions to the O1s signal at binding energies around 533.0 eV (FERREIRA-NETO et al., 2019; TORNOW et al., 2005), the O1s signal contributions related to oxide, hydroxide, or carbonate-related bulk and surface species, in case of tita- nia substrates, are characterized by binding energies around 530.2 and 531.6 eV (±0.1 eV), respectively (FERREIRA-NETO et al., 2019). This facilitates the spectroscopic char- acterization of the chemical environment of oxygen species in thin adsorbates of complex oxygen-containing amphiphilic adsorbates (like ODPA or DMOAP) on titania substrates. 3.3.1 XPS characterization 3.3.1.1 Bulk sample Pure bulk material ODPA is composed of octadecylphosphonic acid molecules with a molecular structure displayed in Fig. 5; the molecule presents the following stoichiometry: C18H39O3P. If a sample of this pure bulk material has a homogeneous composition, the atomic concentrations listed in Table 2 are expected based on this stoichiometry. Additionally, the measured atomic concentration (given in at% ) of carbon is slightly higher than that displayed. The values obtained for carbon, oxygen, and phosphorous are close to the stoichiometrically expected values. The concentration ratios [C]/[O], [C]/[P], and [O]/[P] indicate that a minor excess of carbon species is found at the sample surface, as shown in Table 3. High resolution XPS spectra for the investigated bulk ODPA sample are presented in Fig. 6. C1s high resolution spectra were fitted using two contributions: one contribution at 285.0 eV from aliphatic carbon and the second centred at 285.8 eV due to C*–P species. 42 Table 2 – Comparison between expected for homogeneous solid ODPA and obtained values from the XPS-based evaluations of atomic concentration [C] [O] [P] [Si] Atomic concentration (at% ) Total [C*-C/C*-H] [C*-P] Total [P-O*-H] [P=O] Total Total [Bulk Si] [SiO*2] Expected value 81.92 77.33 4.59 13.64 9.09 4.54 4.54 0 - - Obtained from data evaluation 82.68 78.45 4.23 11.75 7.84 3.90 4.22 1.34 1.18 0.16 Source: Developed by the author From the molecular structure, the intensity for XPS signals related to C*–P and P*–C species in ODPA is expected to be equal to the atomic concentration [P] of phosphorus species in ODPA (4.5 at% ). Based on an appropriate calibration of the applied relative RSF (relative sensitivity factors) for C1s and P2p in the XPS measurement, an area constraint was set to guarantee that the evaluated [C*–P] atomic concentration was equal to the evaluated atomic concentration [P] of phosphorus species in the investigated ODPA bulk material. Table 3 – Ratios between the atomic concentrations of carbon [C], phosphorous [P], and oxygen [O] species, as expected from stoichiometry and found in XPS investiga- tions Atomic concentration ratio [C]/[O] [C]/[P] [O]/[P] Expected from stoichiometry 6.0 18.0 3.0 Found by XPS 7.0 19.6 2.8 Source: Developed by the author Figure 6 – XPS high resolution spectra for investigated bulk sample ODPA, (a) C1s, (b) O1s and (c) P2p signal. Source: Developed by the author Outcomes of the proposed fit agree with the signal positions reported by (MILOšEV; METIKOš-HUKOVIć; PETROVIć, 2012), who reported C*–P species with a C1s binding energy between 285.8 and 286.2 eV in ODPA on a NiTi alloy surface. The obtained atomic concentration ratio [C*–C|C*–H]/[C*–P] equals 18.5, and the expected value is 17. The atomic concentration [C*–C|C*–H] of aliphatic carbon species obtained from the fitting is slightly higher than the expected value. The aspects to assess this difference are the presence of additional substances other than ODPA, a non-precise relative sensitive factor ratio of P2p and C1s, and the presence of non-isotropic dried ODPA micelles on the sample surface inspected by XPS. O1s spectra were fitted using two contributions based on the obtained binding energy values. The causative oxygen species were identified as P=O* (531.6 eV) and 43 P–O*H with minor contributions from SiO*2 (533.0 eV). The positions agree with previous studies of ODPA on different surfaces (THEILE-RASCHE et al., 2020; MILOšEV; METIKOš- HUKOVIć; PETROVIć, 2012; GOUZMAN et al., 2006). The expected and obtained atomic concentration ratio of [P–O*–H]/[P=O*] are both equal two (see Table 2). These results evidence an agreement between the ODPA stoichiometry and the proposed fit. The Si2p signal showed the spin–orbit coupling (∆E = 0.6 eV) from the silicon wafer used as substrate and a peak centred at 103.3 eV from Si*O2 on substrate surface (see Fig. 32a) (GOUZMAN et al., 2006; MOULDER, 1993). Based on the fitting, merely 0.16 at% of silicon is in the form of Si*O2. P2p high resolution spectra displayed only the characteristic spin–orbit coupling with an energy distance of 0.93 eV (CICCO et al., 2019; ROJO et al., 2016). The single P2s peak was centred 57.8 eV higher than the P2p3/2 signal, in agreement with the value reported in the literature (see Fig. 32b) (MOULDER, 1993). Concerning the second amphiphilic agent, pure bulk material DMOAP is composed of DMOAP entities, with the stoichiometry of C26H58ClNO3Si (see Fig. 5). If a sample of such pure bulk material has a homogeneous composition, the atomic concentrations listed in Table 4 are expected in an XPS investigation based on this stoichiometry. To compare the former with a thick vacuum-dried film, the atomic concentration for a fully hydrolysed DMOAP is also listed. Table 4 – Atomic concentration in at% of the distinct surface species by XPS investigations, compared to stoichiometrically expected values. [C] [O] [N] [Si] [Cl] Sample name Total [C*-C/C*-H] [C*-N+] [C*-O-Si] Total Total [-N+] [N*-O] Total Total Bulk DMOAP, vacuum-dried 82.21 56.17 14.30 11.74 9.53 3.06 2.61 0.45 2.80 2.40 Expected value for bulk DMOAP 81.52 59.37 12.50 9.38 9.36 3.13 3.13 - 3.13 3.13 Water exposed DMOAP 80.28 61.33 10.27 8.68 8.73 3.46 3.42 - 4.31 3.22 Expected values for fully hydrolysed DMOAP 79.31 65.52 13.79 0 10.34 3.45 3.45 - 3.45 3.45 Source: Developed by the author For the water treated DMOAP, a hydrolysis of the methoxysilane groups might be expected according to the established behaviour of methoxysilanes with short alkyl chains (SALON; BELGACEM, 2011), as schematically presented below: [(CH3O)3Si(CH2)3N(CH3)2(CH2)17CH3]Cl + 3H2O→[(OH)3Si(CH2)3N(CH3)2(CH2)17CH3]Cl + 3CH3OH This equation is valid for a complete hydrolysis without subsequent condensation. Notably, the process modifies the atomic concentration of carbon in the molecule, and the concentration ratio [C]/[N] can be used as a stoichiometry-based indicator. The [C]/[N] value expected for pristine DMOAP is 26, and for homogeneously composed fully hydrolysed DMOAP is 23. The respectively observed values are 26.9 and 23.2. Details about the bulk and water exposed DMOAP are presented in the C1s high-resolution spectra in Fig. 7 and 34, in the ESI. 44 Figure 7 – XPS C1s high-resolution spectra for DMOAP, (a) bulk, (b) water exposed, and (c) heated blot Source: Developed by the author The binding energies of signal contributions from expected species in C1s spectra for bulk DMOAP were adjusted based on the carbon covalent binding neighbourhoods and considering the suggested binding energy ranges of the expected binding neighbourhoods as compiled by Beamson and Briggs (BEAMSON, 1993). Following this, three distinct binding energies for C1s signal contributions were considered, corresponding to aliphatic C*– C/C*–H (285.0 eV), quaternary ammonium-related C*–N+ (286.0 eV), and C*–O in methyl ester groups (286.8 eV) (see Fig. 7). Significant signal contributions from possible carbon species with binding energies higher than 287 eV were not detected. For a completely hydrolysed DMOAP material, a conversion of the C*–O groups is expected, coinciding with a decrease of the atomic concentration of C*–O from the stoichiometrically expected value 9.38 at% or the measured value of 11.74 at% (non- hydrolysed DMOAP) to 0 at%. However, for [C*–O] of the water-exposed sample, a value of 8.38 at% was found. The minor changes observed for the surface composition suggest a partial hydrolysis caused by water exposure of the DMOAP material after evaporation of the alcoholic solvent. Similarly, the XPS obtained for the thick DMOAP film dried and heated at 100°C in air found small effects on the material composition and, notably, a slight decomposition of methoxy species resulting in a decrease ofthe [C*–O]/[C*–N+] concentration ratio as presented in Table 5. Table 5 – Concentration ratios obtained from fitting C1s signals of DMOAP bulk samples prepared following different processes. Sample name [C*-O]/[C*-N+] ratio Vacuum-dried from solution 0.8 Water exposed 0.8 Thick film after conditioning at 100°C in air 0.6 Source: Developed by the author Subsequently, structural implications of the spectroscopic outcomes will be high- lighted. Considering the stoichiometric composition of DMOAP, hydrolysis is expected to be revealed by decreasing the [Si–O*]/[Si*–O] concentration ratio starting from a value of three. 45 The measured value for bulk DMOAP agrees with this expected value. After the water treat- ment, the value reduced to two, indicating a partial oligomerization of DMOAP molecules due to a condensation following the partial hydrolysis of the methoxysilane moieties. However, the C1s peak shape analysis indicates that a substantial portion of the methoxysilane groups were not hydrolysed. This finding implies that the chemical inertness of the reactive head group of am- phiphilic DMOAP is attributed to the formation of DMOAP micelles in water and to the sterically restricted accessibility of methoxysilane groups for water molecules. Head groups inside micelles may be screened from water by the aliphatic octadecyl tails. Similarly, the for- mation of ordered structures upon hydrolysis of C12–C18 triethoxy(alkyl) silanes was reported by (SHIMOJIMA; SUGAHARA; KURODA, 1997). Even in acidified solution of ethanol-D6 and deuterated water CD3CD2OD : D2O, 80 : 20 w/ w, trimethoxysilanes without amino groups and especially tri-methoxy (7-octen-1-yl) silane (OEMS) required several hours to present significant hydrolysis, as (SALON et al., 2008) reported. A covalent attachment of methoxysilane groups to a titania surface requires the formation of a Si–O–Ti bond starting from a hexagonally coordinated Ti centre. Although the formation of Si–O–Ti bridges is established starting from tetrahedrally coordinated tetraalkoxy compounds under sol–gel conditions, the evidence for the formation of Si–O–Ti bonds starting from titania surfaces is based on hydrosilanes or chlorosilanes rather than alkoxysilanes (FERREIRA-NETO et al., 2019; GUNJI; KASAHARA; ABE, 1999; REN et al., 2008). Starting from a long-chain alkyltrialkoxysilane, such as octadecyltriethoxysilane (ODS) on zinc oxide surfaces, the formation of Si–O–Si was reported to dominate the formation of Si–O–Zn bonds, leading to a cross- linked siloxane network that adheres on the substrate surface via hydrogen bonding or with few anchoring points along the surface (TORUN et al., 2017). Interestingly, acidic properties of both zinc oxide and titania are related to Lewis acid surface sites rather than to Bronsted sites (REN et al., 2008; LIU; TABORA; DAVIS, 1994). The complexation of the acidic substrate surface sites with oxoanions or esters may be driven by Lewis basic electron pair donor properties of the respective adsorbent providing the oxo-bridge. 3.3.1.2 Adsorbed layers on smooth titania substrates High-resolution XPS spectra of sputter-deposited titanium dioxide are displayed in Fig. 8. The Ti2p spectrum was characterised by one doublet showing a B.E. of 458.5 eV for the Ti2p3/2 peak, as expected for titania (MOULDER, 1993). The O1s signal presented two contributions attributed to oxidic oxygen from the titanium dioxide centred at 529.9 eV and organic oxygen/hydroxyl groups at 531.6 eV (THEILE-RASCHE et al., 2020; LANDOULSI et al., 2008). The C1s signal presented four contributions, aliphatic carbon at 285.0 eV, and further contributions at 286.6 eV and 288.7 eV that are also attributed to adventitious carbon 46 in a sorption layer. Finally, in view of the nitrogen species accompanied by an N1s signal with a binding energy of 400.1 eV, a minor contribution of moieties containing organic nitrogen at 285.9 eV was considered (THEILE-RASCHE et al., 2020; LANDOULSI et al., 2008). Figure 8 – XPS high-resolution spectra for sputtered titanium dioxide, (a) Ti2p, (b) O1s, (c) C1s and (d) N1s signal. Source: Developed by the author 3.3.1.2.1 ODPA/titania films Films assembled on these substrates from clear ODPA formulations were used as reference for a well-ordered monolayer with their structure widely studied over the years on polar oxide surfaces like alumina, silica, or titanium aluminium alloys (THEILE-RASCHE et al., 2020; GOUZMAN et al., 2006; HOQUE et al., 2006). Theile-Rasche et al. recently reported photoelectron spectroscopic peculiarities in C1s spectra of ODPA SAMs on Ti0.5Al0.5N hard coatings. Therefore, a more detailed approach for the XPS signal fitting was pursued. As a reference for the C1s and O1s signals of ODPA-based assemblies, the spectra obtained for bulk ODPA were used. The correspondingly fitted signals of the most relevant studied film samples are displayed in Fig. 9, and further findings are presented in Fig. 34. For the adsorbate obtained after 4 h immersion and subsequent rinsing with ethanol, the C1s signal displayed in Fig. 9a was dominated by the contribution from aliphatic carbon species (with a 47 binding energy of 285.0 eV) and showed a contribution provided by C*–P bonded carbon atoms. Figure 9 – High-resolution C1s and O1s XPS spectra obtained for ODPA layers on titania after immersion in ODPA solution for 4 h (a) and (c); 24 h (b) and (d). Source: Developed by the author Comparing the respective spectra obtained for sputter-deposited titania and for the TiO2/ODPA films prepared by 24 h immersion and subsequent rinsing with ethanol (Fig. 9b), there is clear reduction of the carboxylate adsorbed on the surface. However, indications for adventitious carbon species like C*–O (at 286.6 eV) and carboxylates (at 288.7 eV) were still observed as well as a contribution of organic nitrogen at 285.9 eV (THEILE- RASCHE et al., 2020; LANDOULSI et al., 2008). The O1s signal presented two contributions with the peaks centred at 529.9 eV and 531.6 eV (Fig. 9c and d). This indicates a partial competitive desorption of adventitious carbon moieties due to the formation of specifically adsorbed ODPA films. However, a thorough replacement of adsorbed carboxylate species was observed only after considerably longer immersion periods. In detail, when the immersion time was increased from 4 h to 24 h, a significant raise of the ODPA coverage on titania by approximately 70% (as shown in Table 6) was found; however increasing the immersion time to 72 h did not result in a further raise of the ODPA surface coverage. Additionally, the increase of the layer thickness reached a maximum value of 1.5 nm, consistent with a monolayer formation (MCINTYRE et al., 2005). Along with the compaction of the ODPA layers when extending the exposure in ODPA solution to 24 h, the C1s signal became 48 wider and asymmetric, displaying additional features, as depicted in Fig. 9b. These spectral features, as shown in Fig. 9b, may be related to vibrational shake-ups. This observation is consistent with the formation of a self-assembled monolayer structure, as reported by (THEILE-RASCHE et al., 2020) who suggested components at 285.4, 285.8, and 286.4 eV due to vibrational shake-ups similar to the ones reported for polyethylene. Table 6 – Normalized XPS signal intensity ratios for ODPA samples adsorbed on titania, based on the sample immersed for 24 h in ethanolic ODPA solution and then rinsed with ethanol (subsequently labelled “SAM”), and estimated layer thickness Sample name {[P]/[Ti]}/{[P]/[Ti]}SAM {[C]/[P]}/{[C]/[P]}SAM Estimated layer thickness (nm) TiO2/ODPA 4 h EtOH 0.58 0.47 0.4 TiO2/ODPA 24 h EtOH 1.00 1.00 1.5 TiO2/ODPA 72 h EtOH 1.00 0.71 1.3 Bulk ODPA - 0.71 - Source: Developed by the author In contrast to ODPA adsorbates rinsed with ethanol, the film surfaces of equally immersed but water rinsed ODPA/titania specimens presented a signal shape similar to the investigated ODPA bulk sample indicating the presence of physisorbed ODPA species on the surface of the assembled layer. Following the applied XPS-based strategy for assessing details related to the bond- ing situation of the adsorbed species, the O1s spectral region was investigated in more detail. Aside from the two peaks fitted to encompass the contributions in case of the pris- tine titania, the O1s high-resolution signals obtained for thicker water-rinsed ODPA films revealed a further spectral and, thus, chemical feature contributing to a third peak at a higher binding energy around 532.7 eV, as presented in Fig. 9c and d. The concentration ratio [P=O*]/[P–O*H] did not agree with the value of 0.5 found for bulk ODPA. This finding may be due to remaining water trapped close to the polar titania substrate surface or to contributions from the phosphonate species in the underlying ODPA/titania monolayer. Both discussed samples presented a peak at 530.0 and 530.1 eV due to oxidic oxygen species from titania, which indicates that contributions from the film/substrate interphase were also recorded. The second contribution centred at 531.1–531.4 eV is attributed to organic oxygen, P=O* and P–O*–metal ion bonds (THEILE-RASCHE et al., 2020; MILOšEV; METIKOš-HUKOVIć; PETROVIć, 2012; LANDOULSI et al., 2008). In prior studies of films formed from phosphonic acids, contributions resulting in O1s peaks with higher binding energies than 532 eV were tentatively attributed to unbonded phosphonic acid P–O*H groups (THEILE-RASCHE et al., 2020). A lower surface concentration from unbound groups suggests the presence of physisorbed octadecylphosphonic acid. However, the ODPA/titania film obtained after 24 h of immersion and subsequent ethanol rinsing still exhibited a high-binding energy contri- bution in the O1s spectrum. As AFM investigations of this sample surface did not reveal significant rises, such as patches or physisorbed ODPA micelles (as shown in Fig. 12). This 49 finding may indicate that the chemisorbed ODPA is attached to the surface through tri and bi- dentate bounds rather than mono-dentate. Similar XPS findings were not detailed by (THEILE-RASCHE et al., 2020), which may be due to the intense O1s signal contributions of the mixed oxide layer of the Ti0.5Al0.5N coating they investigated. Due to the predominance of a mono-dentate conformation, a higher contribution of unbound groups may be expected, leading to similar O1s contributions, as observed for the investigated bulk ODPA specimen. The observed P2p3/2 binding energy is consistent with the values reported for immobilized phosphonates in the literature (see Fig. 34 and 35) (ROJO et al., 2016; CICCO et al., 2019). 3.3.1.2.2 DMOAP/titania films The XPS findings for the investigated DMOAP/titania films are summarised in Table 7, displaying the detected elements and the contributions of the attributed chemical species. Table 7 – Atomic concentration in at% of the distinct surface species from XPS investigations of DMOAP/titania films [C] [O] [N] [Si] [Ti] [Cl] Sample name [C*-C/C*-C] [C*-N+] [