Exploring the intimate interactions of selected antibiotics with cyclodextrin and silica-based nanocarriers

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(3) UNIVERSIDAD DE CASTILLA-LA MANCHA INSTITUTO DE NANOCIENCIA, NANOTECNOLOGÍA Y MATERIALES MOLECULARES (INAMOL) Facultad de Ciencias Ambientales y Bioquímica Departamento de Química Física Exploring the intimate interactions of selected antibiotics with cyclodextrin and silica-based nanocarriers. Directores: Boiko Y. Cohen y Abderrazzak Douhal Alaui Memoria presentada por Lorenzo Angiolini para optar al grado de Doctor en Nanociencia y Nanotecnología por la Universidad de Castilla-La Mancha.. Toledo, Julio 2019.

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(5) UNIVERSIDAD DE CASTILLA-LA MANCHA INSTITUTO DE NANOCIENCIA, NANOTECNOLOGÍA Y MATERIALES MOLECULARES (INAMOL) Facultad de Ciencias Ambientales y Bioquímica Departamento de Química Física. D. Boiko Y. Cohen, Profesor Titular de Química Física, y D. Abderrazzak Douhal Alaui, Catedrático de Química Física de la Universidad de Castilla-La Mancha, CERTIFICAN que el presente trabajo titulado “Exploring the intimate interactions of selected antibiotics with cyclodextrin and silica-based nanocarriers”, realizado por Lorenzo Angiolini para optar al grado de Doctor en Nanociencia y Nanotecnología por la Universidad de Castilla-La Mancha, ha sido realizado bajo sus dirección y autorizan la presentación del mismo.. Toledo, Julio 2019. Fdo. Boiko Y. Cohen. Fdo. Abderrazzak Douhal Alaui.

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(9) INDEX Abbreviations Abstract Resumen. Chapter 1. Introduction. 1. 1. Antibiotics: general overview and specific issues. 3. 2. Drug delivery concept. 6. 3. Nanocarriers for drug delivery applications. 9. 3.1. Cyclodextrins. 11. 3.2. Nanoparticles. 14. 3.2.1. Gold. 15. 3.2.2. Silver. 15. 3.2.3. Iron Oxide. 16. 3.2.4. Polymers. 17. 3.3. Liposomes. 18. 3.4. Dendrimers. 20. 3.5. Quantum dots. 21. 3.6. Carbon nanotubes. 22. 3.7. Metal-organic framework. 22. 3.8. Silica-based porous materials. 24. 3.8.1. Ordered mesoporous materials. 24. 3.8.2. Zeolites. 26. 3.8.3. Silica particles. 27. 4. Spectroscopic techniques to characterize drug delivery systems. 29. 5. Objectives. 31. 6. References. 32. Chapter 2. Experimental Section. 51. 1. Materials. 53. 1.1. Solvents and salts. 53. 1.2. Antibiotics. 53. 1.3. Nanocarriers. 54. 2. Loading and release methods. 54.

(10) 3. General Photophysics. 56. 4. Spectroscopy techniques. 58. 4.1. Steady-state UV-Vis absorption spectroscopy. 58. 4.2. Steady-state emission spectroscopy. 60. 4.3. Time-resolved spectroscopy techniques. 61. 4.3.1. Time-correlated single-photon counting. 61. 4.3.2. Fluorescence up-conversion. 64. 4.3.3. Fs time-resolved transient absorption. 65. 5. Fluorescence lifetime imagining microscopy. 67. 6. References. 69. Chapter 3. Results and Discussion. 73. 1. Rifampicin: overview and short summary of the results. 75. 2. Clofazimine: overview and short summary of the results. 76. 3. References. 78. 4. Formation, characterization and pH dependence of rifampicin: heptakis(2,6 di-Omethyl)-β-cyclodextrin complexes. 5. Ultrafast dynamics of the antibiotic Rifampicin in solution.. 81 99. 6. Fluorescence Imaging of Antibiotic Clofazimine Encapsulated Within Mesoporous Silica Particle Carriers: Relevance to Drug Delivery and the Effect on its Release.. 125. 7. Single crystal FLIM characterization of clofazimine loaded in silica-based mesoporous materials and zeolites. 160. Chapter 4. Conclusions. 199. 1. Conclusions. 201. 2. Conclusiones. 203.

(11) Abbreviations DDS. Drug delivery systems. NPs. Nanoparticles. CDs. Cyclodextrins. CPT. Camptothecin. Au NPs. Gold nanoparticles. SPR. Surface plasmon resonance. DOX. Doxorubicin. NIR. Near-infrared. MTX. Methotrexate. Ag NPs. Silver nanoparticles. PLX. Paclitaxel. PHA. Polyhydroxyalkanoates,. PLA. Poly-l-lactic acid. PEG. Polyethylene glycol). PCL. Poly-ε-caprolactone. PLGA. Poly(lactic-co-glycolic acid. FA. Folic acid. QDs. Quantum dots. CNTs. Carbon nanotubes. MOF. Metal-organic framework. SMM. Silica-based ordered mesoporous material. NSAI. Nonsteroidal anti-inflammatory. MSPs. Mesoporous silica particles. GIT. Gastrointestinal tract. FLIM. Fluorescence lifetime imaging microscopy. DCM. Dichloromethane. Rif. Rifampicin. CLZ. Clofazimine. DIMEB. Heptakis(2,6-di-O-methyl)-β-cyclodextrin. IC. Internal conversion. VC. Vibrational cooling. ISC. Intersystem crossing. S1. Fundamental state. S1. Singlet state. T1. Triplet state. IRF. Instrumental response function.

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(13) Abstract The aim of this thesis is to investigate the behavior of the antibiotics rifampicin and clofazimine in aqueous solutions and explore their interactions with dimethyl-β-CD and silica nanoporous materials (MCM-41, SBA-15, zeolites, microparticles). The resulting formulations have been studied by means of steady-state (UV-Vis absorption and emission), time-resolved (TCSPC, fluorescence up-conversion, fs-resolved UV-Vis-NIR transient absorption) spectroscopy and fluorescence lifetime imaging microscopy. Chapter 1 (Introduction) presents an overview of antibiotics and recent drug delivery applications for the most studied and promising nanocarriers. It also describes the spectroscopic techniques used for drug delivery systems characterization, focusing on the ones used in this work, and the objectives of this PhD thesis. Chapter 2 (Experimental Section) describes the used materials and techniques to understand the drug-nanocarrier interactions. Chapter 3 (Results and Discussion) shows the obtained results and their discussion in the form of published articles. Here, sections 1 and 2 summarize the characteristic properties of rifampicin and clofazimine, respectively, while the details of each work are found in sections 4, 5, 6 and 7. Section 4 reports on studies of the complexes between rifampicin and dimethylβ-CD at different pHs. Sections 5 elucidates by means of ultrafast time-resolved spectroscopy the photobehaviour of rifampicin in aqueous solutions, where an intramolecular H-bond network plays a key role in the stabilization of the excited state and its relaxation to the ground state. Section 6 reports on the loading, distribution and release of clofazimine from mesoporous silica microparticles. Single particle fluorescence microscopy studies reveal stronger interactions between the antibiotic and the cavities with higher hydrophobicity, which cause slower release rate. Section 7 describes the characterization of interactions and conformation of clofazimine encapsulated in several silica-based mesoporous materials, showing the effects of the properties of the hosts on the photophysical behavior of the drug. Chapter 4 contains the conclusions on the most important information provided by this work. Each chapter will have its own bibliography at the end of the unit except for Chapter 3 where they can be found in section 3..

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(15) Resumen El principal objetivo de esta tesis doctoral es elucidar el comportamiento fotodinámico de dos antibióticos (Rifampicin y Clofazimine) en disoluciones acuosas y determinar cómo interaccionan con una β-ciclodextrina dimetilada y materiales mesoporosos de sílice (MCM41, SBA-15 zeolitas y microparticulas). Los sistemas han sido estudiados por medio de técnicas espectroscópicas de estado estacionario (absorbancia y emisión UV-Vis), resultas en el tiempo (TCSPC, suma de frecuencia con resolución de fs y UV-Vis-IR absorbancia transitoria con resolución de fs) y bajo un microscopio de fluorescencia. El Capítulo 1 (Introduction) presenta una visión general de los antibióticos. Teniendo en cuenta su principal aplicación, transporte y liberación de fármacos, también se describen los principales materiales donde se encapsulan estos fármacos, y las técnicas espectroscópicas que pueden caracterizar dichos sistemas. El Capítulo 2 (Experimental Section) enumera los materiales y describe las técnicas utilizadas en esta tesis doctoral. El Capítulo 3 (Results and Discussion) muestra los resultados obtenidos y las discusiones relacionadas en forma de publicaciones. En este capítulo, las secciones 1 y 2 resumen las propiedades características del Rifampicin y Clofazimine, respectivamente, mientras que los detalles de cada artículo se encuentran en las subsecciones 4, 5, 6 y 7. La sección 4 presenta los estudios de los complejos entre Rifampicin y una β-ciclodextrina dimetilada a diferentes pH. La sección 5 aclara por medio de espectroscopia ultrarrápida resuelta en el tiempo el comportamiento fotofísico del Rifampicin en disoluciones acuosas, donde se ha descubierto que una red de enlaces intermoleculares de hidrogeno es de vital importancia en la estabilización del estado excitado y de su relajación al estado fundamental. La sección 6 presenta la encapsulación, la distribución y la liberación de Clofazimine en micropartículas mesoporosas de sílice. Estudios de FLIM de partículas individuales revelan que hay interacciones más fuertes entre el antibiótico y el sistema que tiene las cavidades más hidrofóbicas, lo que conlleva a una liberación más lenta. La sección 7 describe la caracterización de las interacciones y de la conformación del Clofazimine encapsulado en diferentes materiales mesoporosos de sílice, mostrando los efectos de las propiedades del host sobre el comportamiento fotofísico del antibiótico. El Capítulo 4 contiene las conclusiones más importantes dasarrolladas en este trabajo. Cada capítulo tendrá su propia bibliografía al final de la unidad excepto por el capítulo 3 donde se encuentran en las sección 3..

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(17) Chapter 1. Introduction. 1.

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(19) Chapter 1. Introduction 1. Antibiotics – General Overview and Specific Issues Antimicrobial agents have been included in human history since centuries ago as part of traditional/alternative medicine, but only since the discovery of penicillin in 1929 by Fleming, they started to significantly and systematically improve human life standards [1,2]. The discovery of penicillin was the last step in the process that gave birth to the modern “antibiotic era”. At the beginning of the last century Paul Ehrlich proposed the concept of “magic bullet”: molecules that could actively target only microbial pathogens without damaging the host organism [3]. This concept brought to the development of a systematic screening program on a large-scale between 1904 and 1909. After more than 600 tested organoarsenic derivatives, finally lead to the discovery of an effective antibiotic against syphilis, which was almost incurable before this time [4]. This approach became the standard in the pharmaceutical research for new drugs, antimicrobial agents included, positively resulting in thousands of them identified and introduced in clinical use. Figure 1 shows a timeline of the discoveries of several class of antibiotic. The discovery of penicillin was obtained by Alexander Fleming using a screening method that consisted in inhibitions zones in lawns of pathogenic bacteria on the surface of agar-medium plates. This method has been widely used by researchers to find microorganisms capable of producing antibiotics [5]. The mass production and distribution of penicillin in 1945 made it possible to effectively face for the first time infections caused by Gram-positive bacteria (e.g. Staphylococcus, Streptococcus and Mycobacterium tuberculosis) [6]. After the introduction of penicillin several new natural scaffolds have been discovered upon modification of the specialized metabolism of bacteria and fungi, and new derivatives have been obtained by synthetic modification of the original drugs [1]. Following this golden era, the antibiotics revolutionized the practice of medicine. It became possible to perform invasive surgeries, immune-system shattering chemotherapy, organ transplantation, replacement of joints, corneas and burnt skin, improving quality of life and life expectancy as we know today. However, antibiotics have been only one of the main challenges that microbes have been facing during their existence on this planet, where their flexibility and adaptivity allowed them to live even in extreme condition as from freezing to boiling waters and extreme pHs or temperatures[2].. 3.

(20) Chapter 1. Introduction. Figure 1. A brief overview of classes of antibiotics [7].. 4.

(21) Chapter 1. Introduction Thus, bacteria have been able to develop mechanisms to resist the threat posed by the antibiotics [8]. For example, in some cases they can either modify themselves by replacing and mutating the site targeted by the antibiotics or add layers of protection to it [9]. Additionally, they can fight back altering the antibiotic instead, which can be pumped out of the cells, modified through several processes that lower their efficiency or destroyed by enzymatic reactions. Some major antimicrobial resistance mechanism are represented in Figure 2. These strategies can severely affect the effectiveness of the “magic bullet” approach, as the antibiotics lose their target or begin to face multiple barriers before it may reach it. As soon as resistance mechanism started to occur a race begun [1,4], contraposing the ability to modify and improve the antimicrobial agents efficiency against the promptness of the microbes to adapt to the new changes. Initially, new antibiotics were obtained upon synthetic tweaking of the original natural compounds, or directly by creation of synthetic versions of the natural ones [1]. This synthetic approach brought excellent improvements in the application of antibiotics, such as avoidance of resistance, reduced doses and enhanced efficacy against a larger spectrum of pathogens.. Figure 2. Major antimicrobial resistance mechanisms utilized by Gram-negative bacteria [10].. However, the thrust to develop new scaffold lost intensity between 1960 and 1990, with little to no advancement that inevitably lead to the reappearance of a wave of resistant strains of bacteria. Despite the negative trend, during this period remarkable advancements in technology were done, such as manipulation of recombinant DNA to produce high yields of desired proteins and high-throughput synthesis to create large chemical libraries [11-13]. Genomic data strongly influenced antibiotic discovery, allowing to identify from thousands of sequenced bacterial genomes new targets for antibiotics that were not yet affected by resistance mechanism [14]. The complex and dense network of chemical and genetic. 5.

(22) Chapter 1. Introduction interactions of antibiotics was studied, revealing that non-antibiotic compounds could enhance the efficacy of these drugs, increasing the chances to overcome the resistance mechanisms. Additionally, it was possible to demonstrate the efficiency of combinatorial approach, where different antibiotics delivered together can provide synergistic effects and offer a valid alternative to traditional antibiotic approach [15]. The menace of resistance has become a serious threat to public health in present day [16,17]. The cases of resistant pathogens increase, also due to antibiotics overuse and misuse, while the process of bringing in the market new antibiotics is barely keeping pace. One of the most promising approaches to resolve the drug resistance is the development of delivery systems that can boost the transport of antibiotics and improve their intracellular targeting to avoid the resistance challenge [17-21].. 2. Drug Delivery Concept Drug delivery systems (DDS) are pharmaceutical formulations where nanocarriers are used to achieve targeted delivery or controlled release of therapeutic agents (e.g. anticancer, antibiotic, inflammatory drugs) increasing their bioavailability and efficacy [22-25]. Commonly, nanocarriers are made of biocompatible and biodegradable materials like liposomes, cyclodextrins, inorganic nanoparticles (NPs) and polymers (Figure 3), which are assembled in nanosized structure and share common required properties [26-28].. Figure 3. Examples of some drug nanocarriers.. Nanocarriers for drug delivery must present increased surface and the ability to enhance solubility of encapsulated drugs and increase the rate of dissolution and oral bioavailability,. 6.

(23) Chapter 1. Introduction which would require reduced amount of drug to be delivered, minimizing potential toxicity and side effects of the treatment [29-31]. Additionally, they must present chemical and structural stability over prolonged shelf-storing time against environmental stimuli (e.g. light, humidity) and should be able to perform targeted and/or controlled release (Figure 4) [32-34].. Figure 4. Beneficial effects of targeted drug delivery system to improve the efficacy of drug for the treatment of malignant diseases.. When designing a new formulation for a DDS several elements must be taken in consideration in the process (Figure 5). Firstly, the drugs need to be loaded into the nanocarriers, which usually happens adding the nanocarriers to a solution containing the drug. The solvent used depends on the properties of both the drugs and the nanocarriers (hydrophobicity and hydrophilicity). Once the interaction between drugs and nanocarriers is established the obtained formulation needs to be separated from the solvent in case an organic solvent has been used. Several methodologies like evaporation (room conditions or under vacuum), filtration, phase transfer (to move the DDS to aqueous solution) and centrifugation can be applied. Secondly, the route of delivery should be evaluated since different bioenvironment can be encountered by the DDS, which need to overcome mucosal or cellular barrier, different pH conditions, pass through circulatory system, cells and tissues and face different cellular uptake processes [18,35]. The main administration routes are parental, oral, buccal, nasal, ocular, transdermal, vaginal, rectal, transdermal and intravenous. Each of these routes will provide the drug with different pharmacokinetics and pharmacodynamics that require specific design of the nanocarriers to improve their delivery. Finally, the target of the delivery requires the nanocarrier to perform specific. 7.

(24) Chapter 1. Introduction interactions, since the drug uptake, the recognition and the defense mechanism vary between cancer cells, bacterial cells and sane tissue and the nanocarrier must be tailored in order to perform optimally in the site where they are needed [32,36].. Figure 5. Schematic representation of the path from drug loading to drug delivery.. For oral route, which comes to be the most important way to delivery therapeutic agents, the properties of the drug-delivery nanosystems become important because for different absorption sites specific condition would be required to obtain an optimal release circumstance. Three major routes for delivery have been identified and discussed in the literature. The first is located in the ileum, where specialized cells perform continuous uptake of material by endocytosis and are then transported through lymph vessels directly to systemic circulation. Nanocarriers designed to be absorbed by the lymphatic uptake can be made directly available into circulation avoiding first step metabolism and the liver [37,38]. The second site is found in the small intestine, where functionalized nanosystems can enhance drugs uptake by active interaction with specific cells found at this site, which are responsible for the residence time and uptake there [37,39]. The last site is the stomach, where drugs degraded by the highly acidic environment are taken into the circulation, here or in the intestine in case of drugs having a low pH-dependent absorption [37]. Nanosystems used for targeted delivery in this site need to withstand the low pH conditions, and require equilibrated mucosal adhesion and penetration for optimal transport [40]. The more targeted a drug is, the more safely can be used in specific conditions and the lower its chance of triggering drug resistance, a cautionary concern surrounding the use of broad-spectrum antibiotics.. 8.

(25) Chapter 1. Introduction 3. Nanocarriers for Drug Delivery Applications A widespread problem affecting the newly developed antibiotics is that a high percentage of the approved ones shows poor pharmaceutical properties. The main issue is related to poor solubility, which in several cases is combined with poor permeability, rapid metabolism, degradation resulting in poor safety and tolerability for the patients [23]. In addition to the direct negative effect on people’s health, these drawbacks can compromise the bioavailability of the antibiotics. In some cases, this can lead to a minimal inhibitory concentration lower than the required to safely kill the bacteria, which increases the probability to develop resistance to the treatment. As the process to obtain new molecules is too demanding in terms of time and costs, a lot of effort has been directed towards finding improved formulations of the existing drugs. Changes in the dosage form, synthetic modification to improve the solubility and design of new routes of administration have been proposed to increase the bioavailability of insoluble drugs [23]. Several approaches have been tested to exploit the pH-dependent solubility of ionisable molecules, such as using salt formulations or pH modifiers [41]. Co-solvents and surfactants have been used to facilitate the solubilisation of non-polar insoluble drugs, while preventing their precipitation upon dilution [42]. Additionally, formulations of amorphous drugs have shown improved solubilisation compared to crystalline ones, as a consequence of a reduced lattice energy [43]. Lately, formulations of DDS have gained ground as a valid approach [18]. Figure 6. shows how the interest in drug delivery systems increased in the last 30 years.. Figure 6. Numbers of publications per year up to 2018 for some of the most studied materials for drug delivery applications. Source Scopus.. 9.

(26) Chapter 1. Introduction The rise of nanotechnology allowed to consistently synthesize stable and well characterized nanomaterials with sub-micron sizes [24]. These nanomaterials can carry drugs molecules to a specific target, thus improving its efficiency and the safety of the process. The nanocarriers present high loading capacity, and effectively increase the drug solubility while achieving targeted releases to specific cells [33]. Additionally, the loaded molecules are protected from degradation and deactivation processes, overall improving the bioavailability of the drugs. The sub-micron-size dimensions allow higher intracellular uptake, contributing to a better adsorption of the antibiotic. This allows to optimise the quantity of antibiotic administrated, thus improving its efficiency whereas the toxicity and side-effects are reduced. The introduction of nanomaterials has allowed the development of stimuli-responsive DDS. These external stimuli can consist of changes in the temperature, pH, light, magnetism, ultrasounds, redox and enzymes, and can trigger the release of the antibiotic in a controlled way. The use of nanocarrier for drug delivery applications has been investigated for several pathologies as reported in Figure 7. Some examples of the most advanced drug nano-carriers are reviewed below more in details to provide an overview on nanosystems used for drug delivery applications.. Nanoparticles Drugs Au. Ag. Fe. Liposomes. Dendrimers. QD. CNT. MOF. CDs. Poly. SiO 2 materials. Anticancer. Yes. No. Yes. Yes. Yes. Yes. Yes. Yes. Yes. Yes. Yes. Antibacterial. No. Yes. Yes. Yes. Yes. Yes. Yes. No. No. Yes. Yes. Anti-inflamm.. No. Yes. Yes. Yes. Yes. Yes. Yes. No. Yes. Yes. Yes. No. No. No. Yes. Yes. No. No. No. No. Yes. Yes. Cardiovascular. Yes. No. Yes. Yes. Yes. Yes. No. No. Yes. No. Yes. Glaucoma. Yes. No. No. Yes. Yes. Yes. No. No. Yes. Yes. Yes. Diabetes. Yes. No. Yes. Yes. Yes. Yes. No. No. Yes. Yes. Yes. No. No. Yes. Yes. Yes. Yes. No. No. Yes. Yes. Yes. Yes. Yes. Yes. Yes. Yes. Yes. Yes. No. No. Yes. Yes. Antihypertensive. Pulmonary diseases Brain. Figure 7. Summary of combinations of several drug groups and nanocarriers studied as drug delivery systems.. 10.

(27) Chapter 1. Introduction 3.1. Cyclodextrins Cyclodextrins (CDs) have attracted a lot of interests for applications in medicine and proved to be successful materials drug delivery applications that have already been implemented in several approved formulations for clinical usage [44,45]. These nanomaterials present high versatility in providing hybrid functionalities, which are required to successfully influence the physicochemical and biological mechanisms in order to reach optimal bioavailability for the transported drugs [27]. The ability to provide multifunctionality is one of the most appreciated features when it comes to the challenge of improving drug-specific nanocarrier formulations for drug delivery applications. The basic structure of the natural occurring CDs consists of a cyclic polymer of 6, 7 and 8 glucopyranose units arranged in a truncated cone shape due to their chair conformation, and are usually referred as α-, β- and γ-CDs. Their external surface is hydrophilic, which grants them good solubility in aqueous solutions, whereas the internal cavity is hydrophobic and shows good affinity with many poorly soluble drugs, which can be encapsulated within [46]. Various derivatives have been obtained and used to improve the physicochemical and biopharmaceutical properties of the loaded drugs and the encapsulation ability of these CDs, granting different degrees of hydrophobicity/hydrophilicity and ionic properties [47-49]. Among the most used derivatives to form inclusion complexes, it is possible to find Hydroxypropyl-β-cyclodextrin (HP-β-CD), randomly methylated-β-cyclodextrin (RM-β-CD), and sulfobutylether-βcyclodextrin (SBE-β-CD) [50-52]. The inclusion complexes are formed when the guest molecules are partially or fully inside the host CDs cavity, which is a more suitable environment for interactions than the aqueous media. The formation of complexes is driven by steric and thermodynamic factors, since the water molecules are forced out of the cavity to give space for the hydrophobic molecule, which can then interact with the internal walls of the CDs through van der Waals forces, hydrophobic and H-bond interactions [53]. Besides improving the solubility of the host drugs, CDs have the ability to protect them from specific and non-specific interactions in the physiological media that may cause early decomposition. Additionally, they can enhance drugs permeability through biological membranes and are able to modulate the rate and site of release, which is important for targeted drug-delivery applications [54,55]. Finally, they are classified as safe materials with reduced, if not absent, excipient related adverse effects or hypersensitivities, which make them ideal drug delivery carriers, compared to other polymeric nanosystems. As the design of the nanosystem becomes important to modulate pharmacokinetics and pharmacodynamics for efficient drug-delivery conditions, CDs have been able to improve the performances of several drugs thanks to their properties. For complexes with CDs increased solubility and stability were observed, along with higher bioavailability and dissolution, and reduced toxicity [56-59], which are all fundamental to increase effectiveness. 11.

(28) Chapter 1. Introduction of drugs and, in case of antibiotics, reduce the appearance of resistance mechanisms. In order to widen the applicability of cyclodextrins for biomedical and drug delivery applications, many nanosystems have been designed, such as amphiphilic CDs, CDpolymer conjugate systems and CD-polyrotaxanes (Figure 8). They are obtained upon conjugations of polymers or functional groups attached through enzymatic or chemical processes to the reactive hydroxyl groups of CDs. Then, electrostatic forces, H-bonding, van der Waals and host guest interactions drive the self-assembling of such systems, which exhibit different physicochemical and drug delivery capabilities [60].. Figure 8. Schematic representation of possible cyclodextrin nanosystems [27].. For example, self-assemblies of parent CDs molecules, their derivatives and drug-loaded CDs complexes can be formed upon spontaneous aggregation in aqueous solutions. The micellar-type aggregates formed have usually a range size of 380-750 nm and are regulated by a dynamic equilibrium where continuous aggregate formation and dissociation takes place and inclusion and non-inclusion complexes co-exist causing instability and variations in structural properties [61,62]. The aggregation behaviour is also dependent on the concentration of the CDs complex. In aqueous solutions, inclusion complexes can be formed between CDs and many drugs, where some lipophilic moiety of the guest molecule is hosted in the CDs cavity without requiring the formation of covalent bonds [63]. The drugs in complex with CDs are in rapid equilibrium with free molecules in the solution and the only. 12.

(29) Chapter 1. Introduction driving forces are usually the release of enthalpy-rich water molecules from the cavity, hydrogen bonding, Vander Waals and charge transfer interactions. Many insoluble drugs (e.g. griseofulvin, piroxicam, sulfomethiazole, carbamazepine, camptothecin (CPT), ibuprofen) showed increased solubility, dissolution and cell permeability upon complexation with CDs, achieving a higher bioavailability [45,64-66]. Another CDs-based nanosystem is formed by amphiphilic CDs functionalized with hydrophilic head and hydrophobic tails that provides non-covalent self-assemblies abilities. Several lipids and moieties can be attached on the hydroxyl groups of the CDs molecules, creating various cationic, anionic, neutral CDs nanoassemblies with different structures and properties, which can load and deliver both hydrophobic and hydrophilic drugs [67,68]. Molecules can be selectively loaded either in the aqueous core of the nanoassembly, in the lipophilic exterior or in the hydrophobic cavity of the CDs, which increase the loading capacity compared to polymeric nanocapsules [69]. In order to address loading issues observed with many polymeric particles, CDs and a polymer have been used as comonomers polymer to design new conjugates for drug delivery applications [70]. This approach confers higher drug recognition and provides multiple binding sites with different surface properties that can be adjusted to form specific inclusion complexes, as the CDs can be absorbed on the particle surface, entrapped within the polymer or incorporated in the polymeric chain. Higher gastric permeability and bioavailability have been reported for PLX-CDs complexes when they were incorporated into poly(anhydride) NPs, compared to NPs formulations alone [71]. A further versatile and biocompatible nanocarrier for oral delivery applications based on CDs are nanosponges, which are formed by cross-linked, highly-branched and porous non-aggregating CDs-polymer nanostystems [72]. These nanosponges are prepared by creating a polymer using primary CDs and carbonyl compounds or organic dianhydrides, and present physicochemical and functional properties that are dependent on the chosen cross-linking polymer and their ratio. The random structural arrangement of the cross-linked groups and the CDs cavities works as pores that provide available sites for incorporation of hydrophilic and hydrophobic drug moieties and inclusion and non-inclusion complexes [73]. CDs-nanosponges showed to increase the plasma concentration of complexed tamoxifen, compared to the free drug, providing higher aqueous solubility and therapeutic efficacy [74].. 3.2. Nanoparticles Several types of NPs have been studied to perform drug release, but in the following paragraphs I will focus on the most extensively studied metallic NPs based on gold, silver, and iron oxide and NPs based on synthetic polymers. Worth of mention are NPs that are based on metallic materials such as copper, zinc oxide, titanium oxide, platinum, palladium. 13.

(30) Chapter 1. Introduction and others, and organic materials such as chitosan, alginate, xanthan gum and cellulose, which attracted also interest [75-90]. These NPs have been studied for bioimaging, biosensor and targeted drug delivery. Silica NPs will be reviewed more in detail in the section concerning silica-based porous materials.. 3.2.1.. Gold. Gold NPs (Au NPs) with different shapes (e.g. spherical, rod-like et al.) can be easily synthesized with dimension ranging from 1 to more than 100 nm. They are characterized surface plasmon resonance (SPR) bands, which depend on the Au NPs size and shape [91,92]. The presence of negative charge allows for further functionalization by genes, targeting ligands and drugs [93], which can be conjugated with Au NPs via ionic or covalent bonding, or by physical adsorption. The conjugated attached can be released under control into specific target via biologically controlled stimuli, such as pH or glutathione, or most commonly by light activation [94]. Light irradiation has been used to successfully release a model hydrophobic drug (extracted from Goniothalamus elegans Ast) from monolayer Au NPs [95], by cleaving of the dinitrobenzyl linker. Doxorubicin (DOX) was also effectively released from DNA-coated Au nanorods upon photothermal ablation for the treatment of metastatic breast cancer [96]. Near infra-red (NIR) light has been used to rupture Au NP capsule-shells for the release of the labelled dextran doping, used as a model of biomaterials such as DNA [97]. Conjugates of Au NPs with drugs can increase the antibacterial efficacies of these molecules. It has been reported that colloidal Au (13 nm) combined to methotrexate (MTX), an anticancer drug, has increased its concentration within the cell, thus improving the antibacterial activity, compared to MTX alone [98]. An increase in intracellular DOX concentration inside acidic organelles was observed upon triggered release from 30 nm Au NPs through a pH-sensitive linker [99], while Au nanorods conjugates with SiRNA were reported to efficiently deliver the SiRNA inside neuronal cells [100].. 3.2.2.. Silver. Similar to Au NPs, the silver NPs (Ag NPs) present adjustable size and shape, with enhanced electrical and optical properties, including SPR bands [75]. They can stabilise surface-bound nucleic acids, provide high-density surface ligand attachment, antibacterial properties and show the ability to perform transmembrane delivery and confirmation via imaging [101,102]. Despite their antimicrobial activity, very few studies have been carried out using Ag NPs as carrier for DDS. Functionalization of Ag NPs surface with specific targeting molecules or biocompatible and biodegradable polymers can provide active targeting for molecular recognition, facilitating the uptake of drugs in the cells [103]. For example, Ag NPs were shown to deliver both DOX and alendronate simultaneously to. 14.

(31) Chapter 1. Introduction cervical cancer cells (HeLa), showing permeation of the cell by the drugs-conjugated NPs and increased anticancer activity than DOX or alendronate alone [104]. It has been reported that the antitumoral drug 9-aminoacridine (9-AA) adsorbed on Ag NPs presents controlled release in Hela cells due to the presence of the NPs, which can hold the surrounding 9AA molecules to the surface until a monolayer is formed. Ag NPs contributed to slow down the antiproliferation effect on the cells at low concentration of 9AA and provided the advantage to obtain more spectral information, such as SERS spectra [105]. Positively charged clusters of Ag NPs acted as a bridge between various antibiotics and a magnetite nanocomposite, allowing the drugs to be attached via electrostatic interactions, while preserving the magnetic properties of the nanocomposite [106]. A high bactericidal activity of attached antibiotics rifampicin and doxycycline has been observed during Antimicrobial test with B. pumilus. A successful conjugation of tetracycline and vancomycin with Ag NPs has been reported to present improved antibacterial efficacy thanks to the synergistic effect of this nanocarriers and antibiotics [107,108].. 3.2.3.. Iron Oxide. Iron oxide NPs have been widely studied for medical applications such as hyperthermia treatment, targeted drug delivery and bio imaging thanks to their magnetic properties in addition to the common ones shared by other nanomaterials (e.g. S/V ratio, shape design, simple synthesis etc.) [77]. The most common form used for magnetic particles is a core magnetite (Fe 3 O 4 ) as a mix of oxide of bi- and trivalent iron, which is covered by biocompatible materials that generally are polymers, silica or gold. These are used to protect the formed NPs as oxidation of magnetite tends to alter its paramagnetic abilities. For drug delivery strategies, upon administration, the magnetic particles can be guided by a magnetic field into the bloodstream toward the target site and, once it has been reached, they can release the loaded drug by enzymatic, pH, or temperature activation. Magnetic targeting resulted effective in a study using a transferring-conjugated magnetic silica PLGA NP loaded with DOX and paclitaxel (PLX) for treatment of malignant brain glioma. Mesoporous silica loaded with DOZ was used as coating of a superparamagnetic core of iron oxide. This composition was then covered with PLGA, which was functionalized with the plasma transport protein transferrin and loaded with the drug PLX. The functionalization provided enhancement of the transport of the nanosystem over the brain barrier to target the brain glioma cells. In vitro and in vivo tests showed that cellular uptake was increased by the use of a magnetic field and tumour growth was inhibited [109].. 15.

(32) Chapter 1. Introduction. Figure 9. A schematic representation of structure of drug delivery system based on thermally cross‐ linked superparamagnetic iron oxide nanoparticles for the delivery of DOX and imaging in vivo in cancer cells [110].. Highly magnetic zinc-doped iron oxide (ZnFe 2 O 4 ) NPs have been used to deliver microRNA for the treatment of glioblastoma multiforme brain cancer cells [111]. NPs comprising a γ-Fe 2 O 3 core and a silica porous shell were loaded with the model drug rhodamine B and showed a pH-triggered release at pH 5.5. This composition offered the ability to be tracked by magnetic resonance imaging [112]. Thermally cross-linked superparamagnetic iron oxide NPs were loaded with DOX and used for combined cancer imaging and treatment in vivo (Figure 9). The NPs allowed passive targeting of the tumour by magnetic resonance imaging and provided a precise delivery of anticancer drug with sufficient amount for a successful treatment of the tumor [110].. 3.2.4.. Polymers. Polymeric NPs present outstanding properties such as biodegradability, biocompatibility, nontoxicity, prolonged circulation, good tissue penetration via an active and passive targeting that make them promising candidates for drug delivery applications [113]. Synthetic polymers such as polyhydroxyalkanoates (PHA), poly-l-lactic acid (PLA), polyethylene glycol (PEG), Poly-ε-caprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA), are extensively used for the synthesis of NPs, along with natural polymers. These systems provide high biodegradability, biocompatibility and non-toxicity, which can reduce the risk of side effect in prolonged administrations. Additionally, they allow active and passive cellular targeting, with a facilitated tissue penetration and are able to release to specific targets, in a controlled way, the molecules either loaded or attached on their surface. These materials can be tailored (e.g. composition, solubility, crystallinity, backbone stability, hydrophobicity etc.) for specific use in different way of administration and for different drugs in order to optimise the delivery, while protecting the drugs from degradations. They can be synthesized in order to perform release of the cargo upon diffusion, hydrolysis,. 16.

(33) Chapter 1. Introduction enzymatic degradation and their combination. Finally, the surface of polymeric NPs can be tailored with various ligands including small molecules (e.g fluorescent dyes) peptides, proteins antibodies, which provide flexibility and control in the delivery of drugs (Figure 10). Several studies reported improvement of the therapeutic value of various drugs encapsulated in different polymeric NPs [114].. Figure 10. A schematic representation of the structure and possible functionalizations of a polymeric nanoparticle for applications in targeted drug delivery.. For example, cisplatin (anticancer drug) encapsulated in PLGA–mPEG NPs presented prolonged drug residence in blood upon intravenous administration contraposed to a sustained release once the NPs reached the targeted cancer cells, reducing cisplatin toxicity in healthy tissues [115]. Sustained plasma levels and extended delivery of the neuroleptic compound savoxepine were obtained upon encapsulation in PEG-coated PLA NPs [116]. In vivo studies showed that the drug alone would remain at the site of injection after intramuscular and intravenous administration, whereas when loaded in the NPs it would mostly be located in the macrophages. The cytostatic antitumoral drug tamoxifen exhibited significantly increased level of accumulation within tumour cells and extended presence in the systemic circulation when loaded in polyethylene oxide modified PCL NPs, compared to when it was injected alone [117].. 3.3. Liposomes Liposomes are among the most studied carriers for DDS applications. Liposomal formulations have already been industrialized and commercialized for different treatments, concerning antitumor, antifungal and analgesic treatments among others [118]. The typical liposomes are spherical vesicles in the 50-450 nm size range composed of natural and synthetic phospholipids that form one or more lipid bilayers around an aqueous core [119]. Liposomes are biodegradable and biocompatible materials that can transport both. 17.

(34) Chapter 1. Introduction hydrophobic and hydrophilic molecules, increasing their stability. They possess a membrane structure like cell membranes, which allows for easier incorporation in the cell that can improve the absorption of the encapsulated drugs. Anionic, cationic, or neutral phospholipids can be used to form the bilayers of the liposomes, which allows the encapsulation of hydrophobic materials in the lipid bilayer and hydrophilic molecules in the core [120]. Ligands like peptides, carbohydrates and antibodies can be linked to the surface of the liposomes to grant targeting abilities, while additional functionalization can allow their use in imaging applications (Figure 11) [121].. Figure 11. A schematic representation of the different types of liposomal DDS [121].. Structural materials and different functionalization of the liposomes can alter the delivery efficiency too. Phospholipids with different charges and rigidity can either promote or inhibit their clearance by macrophages, affecting the retention time of the liposomes in the bloodstream [122]. Functionalization using PEG on the surface proved to be an effective way to reduce blood stream clearance and reduce the accumulation of liposomes in the liver and spleen [123]. Fatty acidic carboxyl groups can provide electrostatic repulsion that stabilizes the liposomes, while at acidic pH, like in lysosomes, these groups get protonated and cause the liposomes to become unstable, aggregate and release the guest molecules [124]. At characteristic temperatures the membranes of the liposomes can transit from gel phase to liquid crystalline phase, causing disruption of the membranes followed by release of the loaded drugs [125]. Additionally, the liposomes can incorporate polymers that are directly. 18.

(35) Chapter 1. Introduction heat-sensitive, which would cause the rupture of the liposomes structure when the temperature is increased [126]. Thermosensitive and pH-responsive liposomes are additional promising strategies for drug delivery applications. Therefore, liposomes can be tailored to offer the right delivery conditions depending on the site where the drugs need to be released. A formulation of DOX and liposomes functionalized with PEG showed longer circulation time in plasma, enhanced accumulation in tumoral cells and a superior therapeutic activity over unencapsulated DOX [127]. Liposomes tha present liquidcrystalline phase transition a few degrees above physiological temperature have been used to deliver MTX to murine tumours and showed four-fold higher amount of drug delivered when the tumour was heated to 42°C compared to unheated control tumours [128]. It has been reported that liposomes containing the photopolymerizable phospholipid diacetylene were loaded with high concentration of DOX and remained stable in serum, which usually destabilizes the lipid bilayer causing early leakage [129]. The release of entrapped DOX was achieved only after light irradiation, which causes the photopolymerization of the diacetylene phospholipid and the rupture of the liposome structure.. 3.4. Dendrimers The dendrimers are synthetic macromolecules with highly branched tree and globular shape that grants them good monodispersion and solubility. Dendrimers present a high density of surface functional groups, which grants synergistic/multivalent binding in ligand/receptor recognition, compared to the traditional linear or branched polymers [24,130]. Additionally, ligands with different functional groups such as targeting moieties, imaging probes and biocompatible ligands for specific therapies can be attached on the surface. For example, a polyfunctional dendrimer functionalized with folic acid (FA) and fluorescein was reported to allow localization and imaging during the delivery of the anticancer drug MTX in vitro [131]. The dendrimers have attracted high interest from researchers in drug delivery, as the drug molecules can be encapsulated via covalent and noncovalent interactions (e.g. electrostatic, hydrophobic, H-bond). The release of drugs from dendrimers takes place by either in vivo degradation of the drug-dendrimer covalent bonds, caused by enzymes or environment conditions, or by discharge of the drug due to changes in the environment like pH, temperature etc. Limited therapeutic efficacy and side effects have been reported due to non-specific drug release, making it difficult to precisely control the release of drug at the target site [132]. It has been observed that non-covalent interactions can cause burst release before the accumulation of dendrimers at the tumour site, while drugs conjugated via ester bonds are too stable to achieve the desired minimum eﬀective concentration. A solution to this issue has been developing stimuli-responsive DDS where tumour acidity, redox potential, enzyme and hypoxia can trigger the release from the. 19.

(36) Chapter 1. Introduction dendrimers. Furthermore, controlled local drug delivery can be achieved by light, ultrasound and magnetic field stimulation that triggers the release from stimuli-sensitive dendrimers. Several studies report on the release of DOX from dendrimers upon different stimuli-responsive activation. For example, DOX has been conjugated to dendrimers via orthonitrobenzyl (ONB), a light cleavable group, which afterUV light irradiation initialised the release of the drug, followed by a burst release [133,134]. The conjugates show minimal toxicity on the cells in the dark. When DOX was conjugated to dendrimers via specific peptide linkers and collagen, it was selectively delivered to the tumour, thanks to its enzyme response, and was able to kill the tumoral cells and reduce their growth [135-138]. The use of hydrazone as linker granted stability to the DOX-dendrimer DDS at pH 7.4. Once this DDS reaches the tumour, the extracellular acidity (pH 6.5–6.8) triggers the hydrolyzation of the linker, which is faster after it enters the tumour cells due to the acidic environments of endosomes and lysosomes (pH 6.5−5.5). Thus, release of DOX was selectively performed in presence of cancer cells, while no release was observed in the blood stream at physiological pH [139].. 3.5. Quantum dots Quantum dots (QDs) are semiconductor crystals with diameter ranging from 2 to 10 nm that present unique bright and intensive fluorescence emitting up to the near-infrared (>650 nm) range. Additionally, they possess high quantum yield and offer the possibility to size-tune their light emission [140]. QDs have been studied as nano-theranostics platforms, where they can be either the main nanocarrier or the fluorescent labels for more complex architecture.. Figure 12. Release of conjugated gemcitabine from CdSe/ZnS QDs functionalized with targeting ligand CycloRGD and overcoated using an amphiphilic polymer [141].. CdSe/ZnS QD were functionalized using cathepsin B-cleavable gemcitabine, matrix metalloproteinase-9, detachable PEG and a targeting ligand RGD (Figure 12). These. 20.

(37) Chapter 1. Introduction nanosystems showed prolonged blood circulation time, reduced non-specific interactions and were able to accumulate the drug in pancreatic tumour tissue thanks to enhanced permeability and retention effect, and a multistep release process.[141] Ultrasmall (3 nm) ZnO QDs have been loaded with DOX and functionalized with PEG and hyaluronic acid to target glycoprotein CD44 in cancer cells. Intracellular controlled release of DOX was performed under acidic conditions [142]. Anticancer and antibacterial nanoplatforms were prepared loading CdSe@ZnS QDs with quercetin (QE), showing two- to six-fold increase in cytotoxicity against drug-resistant Escherichia coli and Bacillus subtilis compared to QE alone [143]. A theranostic platform composed of a hydrophobic inner core containing ZnSe:Mn@ZnS QDs and a hydrophilic silica outer shell functionalized with amino groups, which provided targeting capabilities, showed increased solubility over sustained release of the loaded anticancer drug PTX [144]. A model for in vivo tumour treatment was used to enhance targeted delivery of 5-fluorouracil (5-FU) using Mn:ZnS QDs encapsulated in chitosan biopolymer and functionalized with FA. The results showed a reduction in tumour size and less events of metastasis compared to just using 5-FU [145]. Mn:ZnS QDs with chitosan have shown increased incorporation and binding ability of the nanodrug delivery vehicle toward folate receptor-overexpressed cells, proving to be good candidates for targeting, imaging, and drug delivering in cancer therapy [146].. 3.6. Carbon nanotubes Carbon nanotubes (CNTs) have emerged as potential nanocarriers as nontoxic nanomaterial with adjustable optical properties and strong ability to get attached to other particles or molecules, allowing further functionalization [147,148]. These materials have high loading capacity, potential for cell penetration and well-studied release mechanism, which is facilitated by the acidic environment in tumours, and allow wider range of functionalization that can improve the degree of control on the release processes [147,149]. Single-walled CNTs, functionalized with PEG and FA, have been used along with DOX to target and accelerate the treatment of breast cancer cells. In vitro dug delivery was performed at lysosomal pH values (pH 4.0), similar to the one observed for tumours, while studies of in vivo release showed accelerated killing of the tumour cells thanks to the combination of DOX and photothermal ablation caused by the CNTs upon laser irradiation (800 nm) [150]. Highly efficient intracellular drug delivery was performed using multiwalled CNTs functionalized with chitosan and a transactivator of transcription (TAT). The complex showed low toxicity, improved water solubility, higher cell penetration and accumulation in tumours [151]. Magnetic multi-walled CNTs loaded with gemcitabine showed higher effective delivery, preventing metastasis as well as impeding the growth of tumours, compared to magnetically activated carbon NPs in the targeted delivery for treatment of. 21.

(38) Chapter 1. Introduction lymph node tumours [152].. 3.7. Metal-organic frameworks Metal-Organic Framework (MOF) are an emerging class of materials that have drown wide interest in many fields for applications like catalysis, sensing, nonlinear optics and potential biomedical applications as DDS [153]. MOF consist in a class of hybrid materials obtained by self-assembly processes of metallic ions or clusters and organic ligands used to bridge them the metal components. The resulting structure is a three-dimensional network characterized by regular porosity. The virtually limitless combinations of metals and ligands allow to assemble structures with versatile architecture, morphology, size and physicochemical properties [154,155]. Their physicochemical properties remain unaffected by further modifications, maintaining uniformity of the obtained MOF. Additionally, MOF have large surface area and porosity, which provide high loading capacity, while the weak coordination bonds provide better biodegradation. A unique behaviour observed for MOF is their ability to adapt the pore opening to the dimensions of the drugs, in order to optimize drug-matrix interactions thus improving the loading capacity while controlling better the release time. Studies on MOFs based on iron, zinc and zirconium report on a significant increase in the release time for up to several weeks [156-158]. Furthermore, few reports show how the release can be triggered by several single or multiple stimuli, allowing to control the delivery of loaded molecules upon activation by variation in pH, magnetic field, ions, temperature, light and pressure. For example, Fe 2 O 3 -based MOF exhibited controlled release of ibuprofen over 7 days upon magnetic stimulation, which triggered magnetic separation [159]. The introduction of biphenyldicarboxylic acid to the mixtures during the synthesis of an anionic Zinc-based bio-MOF created an ion-responsive system able to deliver procainamide HCl and control its release through ion exchange upon electrostatic interaction [160]. Promising temperature-controlled release of various drugs may be performed using poly(N-isopropyl acrylamide) as a building block. Below 32° C this polymer presents hydrophilicity and tends to dissolve in water [161], which may cause disruption of the MOF structure and release of the drugs. One- and two-photon light irradiation was used to cause stimuli-dependent release from biodegradable MOF of the anticancer drug topotecan (TPT), resulting in higher efficacy against human pancreatic cancer cell PANC1, compared to the free drug [162]. It has been reported that the drug can be used as a ligand and be directly linked to metal sites as in the case of a MOF prepared using non-toxic iron and nicotinic acid as a linker, which has pellagra-curative, vasodilating, and antilipemic properties [163]. Degradation of the hybrid matrix allowed the released of the nicotinic acid under simulated physiological conditions.. 22.

(39) Chapter 1. Introduction 3.8. Silica-based porous materials 3.8.1.. Ordered mesoporous materials. Since the first reported attempt to use a silica-based ordered mesoporous material (SMM) as DDS (2001), when ibuprofen was loaded and released from MCM-41 [164], this class of nanosystems attracted increasing interest for potential biomedical applications [165]. The primary characteristic of SMM like MCM-41 is a bidimensional hexagonal superstructure constituted by silica or aluminosilicate that contain longitudinal mesopores with diameter ranging from 2 to 50 nm. For MCM-41 the pores diameter is usually between 2-10 nm, while for SBA-15, which present the same hexagonal plane structure, is around 9 nm (up to 30 nm). During the synthesis, a supramicellar scaffold is used to condense oligomers of silicate, which is eliminated by extraction or calcination once the desired structure supported by amorphous silica walls is obtained. The dimensions and chemical nature of the skeleton and cavities can be modulated changing the synthesis conditions, which will consequently determine the physicochemical properties of the SMM and its interaction with the environment and guest molecules [166]. Additionally, the SMM can be doped with metals as titanium, aluminium and zirconium and so on [167,168], which can further modify their physicochemical properties. At pH higher than 9 the silica species that will form the inorganic skeleton are usually negatively charged, whereas at neutral or weakly acidic pH the negative charge is rather small, leaving mainly SiOH groups on the surface of the SMM. In addition to that, the charge of the surfactant creating the supramicellar structure can affect the interaction with the silica precursor and modify the properties of the resulting material. The obtained materials show in general high surface area, large pores volume, higher structural stability than polymeric materials and high density of silanol groups that can be functionalized. These are all important characteristics required for drug delivery applications [169]. Higher surface area exerts a greater molecular retention providing extra interactions between the guest molecules and the silica matrix, causing slower drug release and decreased release kinetics compared to materials with smaller surface area [170]. Additionally, the high free surface energy of SMM can increase the stability of amorphous loaded drug. In this state, the drugs have higher solubility and dissolution rate at cost of lowered thermodynamic stability, which would cause fast recrystallization outside of the nanosystem [171]. Also the pore size contributes to prevent the recrystallization of amorphous drug loaded into SMM due to a confinement effect [172], along with affecting the drug loading and release. It has been suggested that the pores should be at least three times bigger than the guest drugs for optimal condition to load and release molecules from the SMM [173], and any change to the pore size will affect the release rate, which allows to control the delivery of the cargo [174,175]. Native silica has negative electrostatic charges. 23.

(40) Chapter 1. Introduction with uniform zones of great electron density, which would allow only drugs molecules charged positively, with electron deficiency or partial positive charges to have access to the pores. However, upon functionalization the negative electron density of SMM can be changed, allowing to exert totally different electrostatic drug-matrix interactions [166].. Figure 14. Preparation of Zn-SBA-15 for efficient delivery of quercetin at pH 5.5 [176].. Using apolar (hydrocarbon chains) groups to functionalize the SMM surface can increase the lipophilic character of the pores whereas polar groups (-NH 2 , SH, -COOH) would increase the hydrophilic character of the matrix and introduce H-bonding capabilities to the type interactions with the guest drugs molecules. In addition, functionalization can reduce the diameter and volume of the pores or instead it can reduce the access of the solvent to the pore, changing the flow and therefore, the kinetic of delivery. For example, SBA-15 functionalized with octyl and octadecyl groups showed a reduced pore volumes and solvent wettability, creating a controlled-release formulation for antibiotic erythromycin [177]. High QE loading capacity (>40%) was obtained using Zn-functionalized SBA-15 and slower in vitro release at pH 5.5 was obtained, compared to the parent SBA-15 carrier (Figure 14). The modification of MCM-41 pores with aminopropyltriethoxysilane (APTES) provided higher loading capability and stronger retention of loaded aspiring, compared to unmodified MCM-41 , due to strong interaction of the functional group with the carboxylic group of aspirin [178]. The rate of release from MCM-41 supports of various cephalosporins, a class of antibiotics, was controlled upon functionalization of the carriers [179]. A triethoxyvinylsilane linker was used to induce higher hydrophobicity to the surface of MCM-41 and the lack of strong interactions with the drug heteroatoms and polar groups lead to higher release rate. On the other hand, hydrophilic APTES linker induced basic properties causing a much slower release rate, which was further enhanced in presence of steric effects on the diffusion for larger molecules loaded into the pores of MCM-41. A pHtriggered release has been reported for MTX loaded in MCM-41, which presented a pHsensitive polymer (poly4-vinylpyridine) grafted mostly on the pores entrance [180]. The positively charged polymer chains would be found in close state at pH 7.4, whereas at acidic pH they would switch to open state, as consequence of repulsive forces between them and. 24.

(41) Chapter 1. Introduction the acidic media. It has been reported that vancomycin can be released in a controlled way from MCM-41 using CdS NPs to cap the the pores of the mesoporous material [181]. As the linker connecting the CdS NPs to the MCM-41 presents disulfide bonds, when the DDS encounters a disulfide-reducing agent, such as dithiothreitol or mercaptoethanol, the NPs are freed, and vancomycin can leave the pores.. 3.8.2.. Zeolites. Zeolites are inorganic materials with crystalline three-dimensional structure formed by SiO 4 and AlO 4 tetrahedra base building block that arranged in a periodic way form a homogenous network of pores connected by channels on a nanoscale [182,183]. Additionally, the structure of zeolites contains positively charged counter-ions, such as Ca 2 +, Na+ , H+ , K+ or transition metal elements, which are required to compensate for the negative charges introduced upon the substitution of silicon by aluminium, granting unique ion-exchange properties [184,185]. Furthermore, the surface silanol groups can be functionalized for additional type of interactions with the guest molecules [186]. A widely used class of these aluminosilicates materials are the zeolites X and Y, which have a similar faujasite-type framework structure and pore diameter but are characterized by different Si/Al ratios (1– 1.5 for zeolite X and >1.5–3 for zeolite Y), thus resulting in distinctive counter-ions distributions [187]. The faujasite zeolites share the same properties as other porous materials, such as high surface area and pore volume, good biocompatibility, improved stability and the ability to customize their properties and interactions upon simple synthetic modifications, which make them interesting candidates for drug delivery applications [185]. The small pores of zeolites (<1 nm) can limit their applications with many drugs and biological molecules that present bigger sizes, but with smaller guest molecules the defined structure and geometry allow the zeolites to perform size selective adsorption and separation [188]. Additionally, when the size of the pores and the hydrodynamic diameter of the encapsulated drugs are comparable, the release presents a zero-order diffusion kinetic, meaning that the release rate is constant [189]. This provides an additional delivery pattern opposed to multi-phase and burst releases. Few studies report the encapsulation of different therapeutic agents into zeolites for drug delivery applications. For example, nonsteroidal anti-inflammatory (NSAI) drugs piroxicam and diclofenac showed over 90% loading percentage in zeolites X and Y [190]. Dissolution tests in stomach pH environment showed that only 10-20% of the drugs released from the zeolites, which is positive a result as high amount of NSAI drugs released in the stomach can irritate its walls. Another NSAI drug, ketoprofen was loaded into zeolites NaX and derivatives in order to improve its delivery as this drug is characterized by short half-life, low bioavailability, and local or systemic disturbance in the gastrointestinal tract. Less than 10% of ketoprofen was released. 25.

(42) Chapter 1. Introduction in the stomach whereas a double phased release was observed at pH 5.0-6.8, which are the ideal conditions for the treatment of inflammatory bowel diseases [182]. A synthetic CuX zeolite was used to load the antitumoral drug cyclophosphamide providing continual maintenance in the blood of the drug concentration without affecting the intensity of its antitumoral effects [191]. Four dealuminated HY and NH 4 Y zeolites showed a two-stage release of ibuprofen, where the first one is governed by a diffusion process not affected by the amount of Al, whereas in the second stage the release is faster for Si/Al ratio up to 22 and then decreases for Si/Al = 62 [192].. 3.8.3.. Mesoporous silica particles. Mesoporous silica particles (MSPs) have attracted attention as potential excipient with suitable properties for oral drug delivery applications targeting the gastrointestinal tract (GIT) [193,194]. Since the oral route is still the preferable route of drug administration [195], MSPs provide the required stability and properties to control the degradation processes and release their cargo at the right time and at the right place [196]. Additionally, MSPs and amorphous silica in general show no toxicity by oral administration, being analytically indistinguishable from colloidal SiO 2 , which has “Generally Recognized As Safe” status from the US FDA [197]. Controlled nucleation and growth using oligomeric silica species and supramolecular surfactant assemblies allow to synthesize particles with size between 10–1000 nm. The MSPs are obtained after removal of the supramolecular structure leading to opening of the porosity and usually show high specific surface area (600–1000 m2 /g) and a high pore volume (0.6–1.0 ml/g), which are optimal to achieve high drug loading levels. The pores size usually ranges between 2-4 nm, but it was demonstrated that MSPs with pore dimensions as large as 30 nm could be synthesized [198,199], increasing the compatibility with a wider range of molecules including the larger molecules and proteins. The nanometer-sized MSPs show higher versatility and improved cellular uptake that grant higher versatility in drug delivery applications. However, particles larger than 2–3 μm remain unabsorbed in the stomach and remain localized there. Thus, for gastrointestinal drug release the micron-sized particles provide an optima delivery approach, enhancing the concentration of drug released in the GIT while reducing the risks of undesired uptake and distribution in the circulation [200,201]. The surface and the pores of MSPs can be tailored upon introduction of functional groups that can improve the interaction with guest drug molecules and the stability or the cellular uptake, provide imaging and targeting abilities, and offer the ability to perform stimuli-activated release [196,202,203]. For the triggerable release the drug can be covalently linked to the silica support through cleavable bonds, or the MSPs can be prepared with a coating that changes conformation upon variation in pH, redox level, or temperature [202,204-209].. 26.

(43) Chapter 1. Introduction. Figure 15. Schematic depiction of the concept of photodynamic therapy releasing an assembly of folic acid and meso‐tetrakis(4‐carboxyphenyl)porphyrin from mesoporous silica particles [210].. These gatekeepers can keep the drug safe inside the MSPs pores until the specific stimulus activates the keeper that open the pore, obtaining a regulated release of the loaded drug molecules [165]. For example, CPT was loaded in MSPs and released in human cancer cell upon photoactivation, showing that cell death increased with increasing activation time [211]. The controlled release was performed by azobenzene molecules tethered to the. interior walls of the nanopores that once irradiated around their isosbestic point (400–450 nm) have their trans-to-cis and cis-to-trans isomerization activated. The untethered portion of these molecules drives a wagging motion that physically expels the cargo from the confined nanopores of MCM-41 particles. A template based on FA has been used to encapsulate meso‐tetrakis(4‐carboxyphenyl)porphyrin (TCPP) within MSPs. It has been demonstrated that FA and TCPP are released to aqueous solutions as a non-covalently assembly, where the assembling process is directly promoted by the silica material (Figure 15). This DDS showed enhanced photo‐induced mortality in cancer cells, compared with the free FA and TCPP. A pH-triggered delivery has been reported for the selective intracellular release of DOX from MSPs into squamous carcinoma (KB-31) cells to induce cell apoptosis [212]. At the entrances of the pores of these MSPs were attached N-methylbenzimidazole. stalks that at pH 7.4 can bind to β-CDs, which effectively close the entrances. Upon entering an acidifying endosomal compartment at pH < 6, the stalks become protonated and β-CDs is detached, allowing the loaded DOX molecules to be released from the MSPs. In a different MSPs-based DDS, the in vitro delivery of DOX achieved in a breast cancer cell line MDAMB-231 utilizing an oscillating magnetic field to trigger the release [213]. These MSPs present an iron oxide nanocrystal-core that upon the application of a magnetic field starts to. 27.

(44) Chapter 1. Introduction generate heat, which increases the internal temperature of the system and causes the thermosensitive pseudorotaxanes caps to disassemble and free up the entrance of the pores. Minimal drug release was observed when the loaded particles were taken up by the cells, indicating that the DOX molecules were kept inside the MSPs until the magnetic field was applied.. 4. Spectroscopic techniques for drug delivery systems characterization Characterization of DDS is an important step to understand their physical and chemical behaviour in the both at the molecular and biological level. The properties of a material (size, morphology, surface charge, porosity, crystalline arrangement) and the type of interactions with the guest drugs that they transport affect the delivery processes. Thus, characterizing the DDS formulations becomes crucial to control and modify the transport and release properties in order to obtain the optimal conditions for the delivery. Several techniques are used to characterize the properties of the nanocarriers, the interactions with the loaded drugs and the effect of the media, when the sample can be studied in solution [214-225]. These techniques include methods of thermal analysis (e.g. Thermogravimetry,. Differential Scanning Calorimetry and Thermal microscopy) and crystallographic methods (e.g. Powder X-Ray Diffraction, X-Ray Diffraction and Small-Angle X-Ray Scattering). Additionally, spectroscopic methods such as UV-Vis and Infrared Spectroscopy (e.g. Fourier Transform Infrared Spectroscopy, Raman Spectroscopy, Circular Dichroism, Steady-state Absorption and Emission, Fluorescence Up-Conversion and Transient Absorption), along with Nuclear Magnetic Resonance Spectroscopy are used. Finally, Microscopy methods (e.g. Scanning Electron Microscopy, Transmission Electron Microscopy, Atomic Force Microscopy and Confocal Microscopy) are used as well for characterization of DDS. I will focus only on Steady-State and Time-Resolved UV-Vis-NIR spectroscopy and Confocal Microscopy techniques, since are the ones used in this PhD thesis. However, it was important mentioning some of the available methods as each technique provides unique approaches and advantages on the study of the DDS properties and interactions, offering complementary information that can improve the understanding of these systems. Steady-state and time-resolved UV-Vis and NIR spectroscopic techniques are powerful tools to study and characterize the type and degree of interaction between drugs molecules and the nanocavities of DDS. The variation of the photophysical and photochemical properties of loaded molecules due to the host-guest interactions can be measured providing important information on the nanosystem under study [226]. For example, steady-state absorption and emission can be used to provide an initial characterization of the inclusion equilibrium constant and the processes that affect it, such. 28.

(45) Chapter 1. Introduction as H-bonds formation, polarity variation and steric interactions that are dependent on the size matching between the guest and the host cavities [162,227-230]. Additionally, they can determine the stoichiometry of the formed complexes that allows to identify and evaluate the site and the strength of the interaction [229,231-233]. However, photodynamics from the excited state of a molecule are complex and steady-state measurements alone cannot always fully distinguish the photophysical process active in specific conditions [234]. Time-resolved techniques, such as transient absorption and fluorescence up-conversion, with temporal resolution from ns to fs are extremely sensitive to subtle changes in the environment of the studied molecules and offer the opportunity to observer in detail the dynamics behaviour of drugs [226,235]. Such sensitivity to small environmental changes allows to detect twisting motion, vibrational relaxation (cooling), conformational changes, solvation dynamics, isomerization, energy, electron and, proton transfer [236]. Therefore, it becomes possible to observe in real time the effect of the nanoconfinement of the nanocarriers cavities on the physicochemical properties of the encapsulated molecules that can be affected by shielding effect, restriction of the molecular movement, H-bonding and electronic donating/accepting environment [237]. Since fs techniques requires high concentration of the molecules, time correlated single photon counting, a ps to ns spectroscopic technique, allows to maintain a good temporal resolution and a wider window, while reducing the concentration needed to observe a good signal to noise ratio.. Several time-resolved studies report on the. encapsulation of drugs within nanocarriers describing the dynamics of the complexes formed. For example, the complexation of tolmetin, a NSAI drug, by β-CD showed that the decarboxylation of the drug was reduced upon inclusion. Additionally, the incorporation of tolmetin transient intermediates into the hydrophobic cavity slowed down the photodecomposition of the drug [226]. It has been observed that methylation of CD causes stronger hydrophobic interaction that lead to robust host-guest interactions with TPT and affect the excited-state proton-transfer (ESPT) rate constants of this drug [229]. The interaction between doxycycline and oxytetracycline with micellar carriers has been reported showing a pH effect on the inclusion complexes, which present changes in the spectroscopic and dynamical properties [238]. When the drugs are present as zwitterionic molecules they are entrapped in the micelle cavity, while in the monoanionic form they show a strong one-to-one interaction with the positively charged surfactant heads. Fluorescence lifetime imaging microscopy (FLIM), in addition to the direct determination of fluorescence lifetimes (ps to ns) at every spot of an image simultaneously offer the option to qualitatively visualize the relative distribution of these lifetimes in the image, while being able to suppress undesired fluorescence signals. The images obtained can provide further information on the spectroscopic properties and the localization of encapsulated drugs within the nanocavities or during the delivery process within the cells. For example, the formation of 1 : 1 and 1 : 2 complexes of DOX with two citric acid crosslinked γ-cyclodextrin. 29.