Repositorio Institucional de Documentos

Ordered mesoporous silica monoliths: synthesis, preparation and potential applications University of Zaragoza Institute of Nanoscience of Aragón Nanoporous Films and Particles Group Author: María Gracia Colmenares Quevedo Directors: Manuel Arruebo Gordo Francisco Balas Nieto Master in Nanostructured Materials for Nanotechnological Applications Thanks to all the people who helped make this possible. María Gracia CONTENTS Contents I. Introduction and Objectives .................................................................................................. 1 II. Theoretical Background ........................................................................................................ 3 II.1. Mesoporous materials: mesoporous silicas .................................................................. 3 II.2. Bimodal Mesoporous silica monoliths .......................................................................... 7 II.3. Controlled drug delivery ................................................................................................ 8 III. Experimental Methods .................................................................................................... 15 III.1. Mesoporous silica synthesis and functionalization ..................................................... 15 III.2. Monolith preparation and functionalization............................................................... 16 III.3. Mesoporous material and monolith characterization ................................................ 18 III.4. Cefuroxime loading and release .................................................................................. 20 IV. Results and Discussion .................................................................................................... 23 IV.1. Monolith and mesoporous materials characterization ............................................... 23 IV.2. cefuroxime release ...................................................................................................... 29 V. Conclusions ......................................................................................................................... 35 VI. References ....................................................................................................................... 37 II. THEORETICAL BACKGROUND 6 could be achieved by developing new synthesis pathways and by taking advantage of the liquid-crystal chemistry provided by the surfactant. The original approach was extended to the use of triblock copolymer templates under acidic conditions, by which means the Santa Barbara Amorphous (SBA) silica phases were synthesized 7 . The material under study, SBA-15, is characterized by a large BET surface area (>700m2/g) with large pore diameter (~ 8nm) and large pore wall thickness. The large wall thickness results in higher hydrothermal stability than M41S materials 17 . Furthermore, SBA-15 exhibits continuous porous channels and has a characteristic hexagonal structure ( p 6 mm ) 18 . In the case of SBA-15 synthesis, a nonionic triblock copolymer, namely Pluronic (P123) 19 is used as a micelle template. P123 is one of the Pluronics triblock copolymers, which are composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene (see Fig. II.3); its nominal chemical formula is HO(CH 2 CH 2 O) 20 (CH 2 CH(CH 3 )O) 70 (CH 2 CH 2 O) 20 H, which corresponds to a molecular weight of about 5800 Da. Fig. II.3. Molecular structure of Pluronics® triblock copolymers. Because of their amphiphilic structure, these triblock copolymers have surfactant properties that make them useful in many applications. As a result of their amphiphilic nature, surfactants can associate into supramolecular arrays, forming micelles with a configuration that depends on the nature of the solvent (an aqueous solvent, for example, would lead to micelles with hydrophobic centers and hydrophilic tails). The extent of micellization, the shape of the micelles, and the aggregation of micelles into liquid crystals depend on the surfactant concentration. At slightly higher concentrations, called the critical micelle concentration (CMC1), the individual surfactant molecules form small, spherical aggregates (micelles). At higher concentrations (CMC2), where the amount of solvent available between the micelles decreases, spherical micelles can coalesce to form elongated cylindrical micelles (characteristic templates for SBA-15). At slightly higher concentrations, liquid-crystalline (LC) phases form; as concentration increases, rodlike micelles aggregate to form hexagonal close-packed LC arrays and cubic bicontinuous LC phases form followed by LC lamellar phases 20 . The silica source employed for SBA-15 synthesis is the metal alkoxide tetraethyl orthosilicate (TEOS), which is a tetrahedral molecule consisting of four ethyl groups attached to a SiO 44- ion, called an orthosilicate (see Fig. II.4). The nominal chemical formula of TEOS is Si(OC 2 H 5 ) 4 . Fig. II.4. Molecular structure of tetraethylorthosilicate (TEOS). The synthesis of mesoporous silica follows a metal alkoxide-based sol-gel reaction. The sol-gel transition of a metal alkoxide solution is generally caused by two kinds of reactions, i.e., II. THEORETICAL BACKGROUND 7 hydrolysis and polycondensation. Although the former one is necessary for the latter to take place, these reactions are known to proceed in parallel from a very early stage of the whole reaction21. In usual cases, the hydrolysis is initiated by mixing water with alkoxide in the presence of alcohol as a co-solvent (Ec. II.1). The hydrolysis of M-OR produces M-OH which subsequently condenses with other M-OH to produce a polycondensed species containing an M-O-M linkage and water (Ec. II.2). A successive condensation leads to the growth of metaloxane oligomers which subsequently link together to form a gel network (Ec. II.3). In the presence of a limited amount of water, the alcohol producing condensation is also possible. The overall reactions for silicon alkoxide, Si(OR)4 are expressed as follows: ܵ݅ሺܱܴሻସ+ ܪଶܱ → ܵ݅ሺܱܪሻሺܱܴሻଷ+ ܴܱܪ …(Ec. II.1) ܵ݅ − ܱܪ + ܵ݅ − ܱܪ → ܵ݅ − ܱ − ܵ݅ + ܪଶܱ …(Ec. II.2) ܵ݅ − ܱܪ + ܵ݅ − ܱܴ → ܵ݅ − ܱ − ܵ݅ + ܴܱܪ …(Ec. II.3) TEOS has the remarkable property of easily converting into silicon dioxide; a silica network with silanol groups on the external surface is obtained and the side product is ethanol. Silicon alkoxides exhibit extraordinarily slow hydrolysis and polycondensation kinetics compared to other metal alkoxides. For this reason, a controlled hydrolysis of silicon alkoxides is much easier and the silica gel materials can be processed into various morphologies by varying the processing method. The reaction proceeds as was described above, in summary: ܵ݅ሺܱܥଶܪହሻସ+ 2ܪଶܱ → ܱܵ݅ଶ+ 4ܥଶܪହܱ …(Ec. II.4) For the case of SBA-15 synthesis, TEOS molecules are condensed on the hydrophilic surface of cylindrical micelles, ensuring that a faithful replica of the template is obtained. A fundamental condition for these methods of synthesis is that an attractive interaction between the template and the silica precursor is produced to ensure inclusion of the structure director without phase separation taking place. Interactions between nonionic templates (as P123) and silica sources are mediated through hydrogen bonds22. The structure of the wall of the resulting pores consists of a disordered network of siloxane bridges and free silanol groups ; this framework is well suited for the development of bonded selective sorption phases, via functionalization with organic molecules (see section III.1). II.2. BIMODAL MESOPOROUS SILICA MONOLITHS Mesoporous silicas were initially synthesized exclusively as powders, which made them suitable for catalytic and separation applications, with a variety of ordered mesophase architectures available (cubic, hexagonal, lamellar, etc), as was mentioned previously. Since then, however, efforts to improve the processability and broaden application possibilities of these materials have been made, resulting in low-molecular weight surfactant/inorganic mesophase composites and mesoporous solids in the form of films, spheres, etc. Optically transparent silica/block copolymer monoliths have been synthesized with high degrees of mesoscopic order, by means of solvent evaporation within the mesostructure (note that the natural meaning of monolith is a geological feature such as a mountain, consisting of a II. THEORETICAL BACKGROUND 8 single massive stone or rock)23,24. These monoliths, although rigid and free of cracks, are not mesoporous materials, but mesostructured composites as the block copolymer template is not removed after synthesis25. Monolithic silicas with well-defined porous structures have been mainly focused on investigations for applications in separations. HPLC columns based on such materials were first reported in 199626, and are characterized by higher total porosity and permeability compared to packed columns, allowing operation at low pressures, yet at higher flow rates, thus reducing the analysis time drastically. Monolithic silica HPLC columns are already marketed by Merck Co. under the trade name Chromolith™. These monoliths are readily prepared inside HPLC columns, for exclusive use in chromatography. For many applications, such as size selective adsorption27 materials with both small and large pores arranged in a hierarchical structure-in-structure fashion are desirable. So-called bimodal silica monoliths were first synthesized by Nakanishi, et. al. in 2005 by direct synthesis28, resulting in confined monoliths with an ordered hierarchical porous structure. A common problem regarding preparation said structures, however, is that they tend to be mechanically unstable unless encapsulated or embedded in a polymeric network (i.e., monoliths tend to crack upon calcination and removing from the mold, yielding irregular pieces and coarse powders, especially as the diameter of the monolith decreases). Leventis, et. al., prepared polymer-encapsulated bimodal monoliths with increased mechanic stability. The same study includes attempts to prepare monoliths without polymeric encapsulation, proving unsuccessful29. In response to this and due to the important amount of potential applications, various techniques for synthesizing silica monoliths with an ordered macro/mesoporous structure were developed. Bimodal macro/mesoporous silica monoliths can be prepared in two ways: by direct-templating of mesoporous silica in a mold that holds the desired shape of the monolith (dual-templating techniques30), and by using as-synthesized or modified mesoporous silica within a polymeric network that aids in holding the desired shape31. Another preparation method is by controlled fusion of mesoporous spherical silica particles32, yielding mechanically stable monoliths with tunable open macropores and surfactant-templated ordered mesopores. Recent advances in research concerning hierarchically ordered mesoporous silica monoliths are focused mainly on applications in catalysis and as catalytic reactors. These monolith rods were used as continuous flow catalytic microreactors after introduction of the active sites via grafting of organic functions, or via the transformation of the amorphous silica skeleton into other mesoporous silica species. These monolithic reactors demonstrated higher productivities than batch or packed-bed reactors for various model reactions33. II.3. CONTROLLED DRUG DELIVERY In the present study, silica monoliths exhibiting macropores of approximately 3 µm and mesopores ranging from 5 to 7 nm were prepared for testing in controlled drug delivery of an antibiotic as a model drug. II. THEORETICAL BACKGROUND 9 Drug delivery is a key factor contributing to the therapeutic and commercial potential of many drugs and related products. It is the driving force behind the development of many new devices and formulation-based projects. Conventional forms of drug delivery include oral, topical, inhaled, injections, etc. However, more sophisticated delivery systems must take into account pharmacokinetic principles, specific drug characteristics and variability of response - from one person to another and within the same person under different conditions. The therapeutic efficiency of a drug can be enhanced and toxic effects reduced if, for example, the amount and persistence of the drug in the vicinity of the target cells is increased, while reducing exposure of the drug to non-targeted cells. Design of any drug delivery system (DDS) must simultaneously take into consideration factors such as34: ⋅ drug properties ⋅ biocompatibility ⋅ targeting abilities ⋅ nature of delivery vehicle ⋅ mechanism of drug release ⋅ duration of delivery ⋅ route of administration These are the principles behind controlled drug delivery. It is not easy, however, to achieve all these factors in one system because of their extensive independency. Various approaches for controlled drug delivery are briefly described below34: a) Localized drug delivery: It is desired in many cases to deliver drugs at a specific site inside the body, to a particular diseased tissue or organ, especially for drugs that tend to provoke unintended side effects that can be severe (i.e. anticancer drugs, antiinflammatory steroids, etc.). b) Targeted drug delivery: Delivery would typically involve a recognition event between the drug carrier and specific receptors at the cell or tissue surface. The concept of targeted drug delivery differs from localized delivery in the sense that the latter simply implies localization of the delivery vehicle in the vicinity of an organ or tissue site, while targeting is based on delivery to specific cell or tissue types. c) Sustained drug delivery (Zero order release profile): Ideally, the blood level of a drug would remain constant throughout the delivery period, yielding a continuous release profile consistent with zero-order kinetics35. However, injected or ingested drugs follow first-order kinetics, with high blood levels after initial administration (which could result in toxicity) followed by a rapid decrease in blood concentration (resulting in low drug efficacy). This drug release profile is undesirable; advantages of a continuous release include reduced risk of toxic effects, predictable and extended duration of drug action, and reduced frequency of dosing. See Fig. II.5. II. THEORETICAL BACKGROUND 10 d) Modulated drug delivery (nonzero-order release profile): Patterning release profile still remains a great challenge in drug delivery. A delivery system with a manipulable nonzero-order profile would ideally be tunable to each situation. e) Feedback controlled drug delivery: The ideal drug delivery system is the feedback controlled system, consisting of drug release in response to a therapeutic marker. Two classes can be defined: modulated devices, in which monitoring the chemical environment is possible in order to change the delivery rate continuously, and triggered devices, in which no release takes place until it is triggered by a marker or an external stimuli. f) Implantable controlled drug delivery devices: implantable devices are very attractive for a number of classes of drugs, particularly those that cannot be delivered via the oral route or are irregularly absorbed via the gastrointestinal tract. For certain diseases that require chronic administration of drugs, an implantable release device is highly useful. The monoliths developed in this project have a potential use as implantable controlled drug delivery devices applied in traumatology and orthopedic surgery. In these scenarios, bacterial infection after implantation is very common, making antibiotic-delivering implants an attractive option. Moreover, in cases where bone regeneration is necessary, the controlled release of growth factors may enhance the healing process after a fracture. Fig. II.5. Zero-order (b) and first-order (a) kinetic profiles. A zero-order profile is preferable, as only one dose is necessary to maintain constant drug concentration within the therapeutic range for a prolonged period of time. Important parameters to consider in the design of a drug delivery vehicle include 36 : a) use of biocompatible materials with simple robust processes for biomaterial assembly, conjugation chemistry, and purification steps; b) ability to optimize in parallel the numerous biophysicochemical parameters of targeted drug delivery vehicles important for pharmacokinetic control and possible cell uptake; and c) development of scalable unit operations amenable to manufacturing large quantities of targeted drug delivery systems needed for clinical translation. II. THEORETICAL BACKGROUND 11 One of the applications of the present investigation is based on the study of the release of a model antibiotic (namely cefuroxime sodium salt) from mesoporous powder and bimodal monolithic SBA-15, in order to assess differences between morphologies and pure-silica functionalized surfaces. Cefuroxime is a type of antibiotic called a cephalosporin, related to penicillin. First-generation cephalosporins are active predominantly against Grampositive bacteria, and successive generations have increased activity against Gramnegative bacteria. The impact of mesoporous silica as a drug delivery vehicle is described below. Mesoporous silica in drug delivery Mesoporous silica matrices have been investigated as potential drug carriers due to the following features37: a) Ordered pore network: as was previously described, mesoporous silica has a characteristic highly ordered array of pores whose structure depends on synthesis conditions. The ordered mesopore network is therefore tunable very homogeneous in size, allowing fine control of drug loading and release kinetics; b) High pore volume: large drug loads are viable; c) High surface area: implies high potential for drug adsorption; d) Silanol-covered surface: possible functionalization to allow better control over drug loading and release; e) Biocompatibility38. Various studies of release of model drug molecules from mesoporous silica matrices of different pore sizes have been carried out, suggesting that several factors could affect the release profile of the hosted molecule. Studies on release of model drug molecules of different molecular sizes from mesoporous silicas with a cubic pore ordering39 indicated that release tends to become slower as the pore size of the matrix decreases or the molecular size of the drug increases, and release rate was decreased further by functionalizing the mesopore wall (see following section). Comparison studies of release of a model antibiotic from hexagonallyordered mesoporous silica powder and discs40, resulting in release with a higher degree of control than conventional presentations of the antibiotic. It was also observed that release was slower and more controlled from disks than from powders. Organic-modified mesoporous silica The interaction between mesoporous matrices and drugs is also decisive for designing controlled drug delivery systems for clinical applications; the drug-material interaction is determinant regarding the adsorption and delivery behavior of the drug. One of the characteristics of mesoporous silica materials is the presence of a high concentration of silanol groups in the mesopores, which can be functionalized for the control of pore size and surface properties. Reactive and passive organic groups can be incorporated into the solid either by simultaneous condensation of corresponding silica and organosilica precursors (cocondensation), by incorporating organic groups as bridging components directly and specifically into the pore walls by the use of bis-silylated single-source organosilica precursors II. THEORETICAL BACKGROUND 12 (production of periodic mesoporous organosilicas) or by subsequent modification of the pore surface of a purely inorganic silica material (grafting) 22 . In the present study, SBA-15 was modified by grafting, which refers to the subsequent modification of the inner surfaces of mesostructured silica phases with organic groups. This process is carried out primarily by reaction of organosilanes of the type (R’O) 3 SiR; in principle, it’s possible to functionalize with a variety of organic groups by modifying the organic residue (see Fig. II.6). Fig. II.6. Grafting for organic modification of mesoporous silica with terminal organosilanes 22 . It must be taken into account that functionalization involves a decrease in pore size, which can directly affect the release kinetics, especially for drugs with a size similar to that of the pore diameter 41 . Several investigations of drug release from functionalized mesoporous silica have been reported. Functionalization of SBA-15 with long alkyl chains resulted in a decreased release rate compared to as-calcined SBA-15 41 . Postsynthesis amine-functionalized SBA-15 yielded more control of release kinetics than the as-calcined samples: ibuprofen release rates were improved due to presence of ionic interaction between carboxyl groups in ibuprofen and amine groups on the surface of SBA-15, and release of bovine serum albumin (BSA) was improved due to o the balance of electrostatic interaction and hydrophilic interaction between BSA and the SBA-15 matrix 10 . Drug release profiles and kinetics The drug delivery profile is very relevant in the design of a drug delivery system. Release kinetics can be optimized by understanding the release process and determining the release mechanism. For many DDS, the drug release process can be modeled by a dissolution profile. For mesoporous silica, it has been shown that drug release is diffusion controlled 40,42 , making it possible to calculate kinetic parameters for comparison purposes. II. THEORETICAL BACKGROUND 13 Fig. II.7. Common release profiles for mesoporous materials 37 . Fig. II.7 shows common release profiles for mesoporous materials 37 . Profile a corresponds to unmodified surfaces, in which an initial burst release is followed by a slow release; this profile is desirable when an immediate high dose is required, as in acute infections. Profile b is associated with diffusion or dissolution processes, and generally follows first-order kinetics with respect to drug concentration. Profile c corresponds to zero-order kinetics, in which the release process is constant and dependent on time; this kind of profile is desirable for longterm drug delivery systems. Profile d represents a stimulus-responsive system in which external changes (such as pH, temperature, magnetic field, etc.) allow controlled release. III. EXPERIMENTAL METHODS 20 TGA was employed to confirm the presence of APTES and cefuroxime in the sample. Measurements were carried out using a heating rate of 5ºC/min until 900ºC using a N 2 flow of 15ml/min (TA Instruments™ Thermogravimetric Analyzer). III.3.7. UV-Visible spectroscopy UV-Visible spectroscopy (UV-Vis) is a colorimetric technique widely used to identify organic and inorganic species within a sample. UV-Vis was used for two purposes: as a tool to determine the amount of cefuroxime loaded onto SBA-15 powders and monoliths and to study the release mechanisms of said materials. UV-Vis measurements were carried out on an UV-Vis spectrophotometer (8453 UV-Visible, Agilent™) at 278 nm, which is the characteristic absorbance wavelength of cefuroxime. The equipment consists of a light source of two lamps (wavelength range from 190 to 1100 nm), a monochromator to separate different wavelengths and a detector. III.4. CEFUROXIME LOADING AND RELEASE Drug delivery experiments were conducted on four different samples, namely unfunctionalized and functionalized SBA-15 powders (P-SBA-15 and P-SBA-15’) and monoliths (M-SBA-15 and M-SBA-15’, respectively). III.4.1. Cefuroxime loading Loading of cefuroxime sodium salt (CEF, Sigma- Aldrich, Fig. III.6) onto SBA-15 powders and monoliths was accomplished using the same technique for both materials, in order to establish differences regarding the amount of drug adsorbed onto the mesoporous material, specifically due to the difference in macroscopic configuration. Fig. III.6. Molecular structure of Cefuroxime Sodium Salt (CEF). Prior to loading, it is necessary to dehydrate the pores of the material, which is done by lyophilization (Telstar Cryodos Lyophilizer). Previous work carried out by members of the NFP group have demonstrated that loading of cefuroxime in a 1:2 proportion with respect to the mesoporous material yields the highest possible loads; for this reason this proportion was chosen as a base for cefuroxime loading (Carmona and Balas, unpublished data). The corresponding volume of a solution of cefuroxime in DIW with a concentration of 5 mg/ml was added onto approximately 20 mg of SBA-15 powders and monoliths alike immediately after lyophilizing in a glass vial. For monolith loading, the vial underwent moderate stirring (to avoid monolith damage) in an orbital shaker (IKA®KS 130 Control) at 230 rpm and room temperature; for powder loading the vial was III. EXPERIMENTAL METHODS 21 placed on a roller and tilt mixer (SELECTA Movil-Rod). After 24 hours, the powders and monoliths were filtered on laboratory filter paper and washed once with 1ml DIW, and left to dry in ambient conditions for another 24 hours. III.4.2. Cefuroxime release Cefuroxime release mechanisms from SBA-15 powders and monoliths were determined using UV-Vis spectroscopy in a continuous mode (see Fig. III.7). It was possible to measure the absorbance of the release media in order to monitor cefuroxime release. Measurements were carried out at controlled time intervals at the characteristic absorbance wavelength of cefuroxime. Absorbance measurements were carried out in the UV-Vis spectrophotometer described in section III.3.7. Fig. III.7. Continuous-measurement release setup. The setup consists of a circuit comprising polyurethane piping and a peristaltic pump that directs the release media from the initial release vial through a quartz cuvette where absorbance measurements are executed and back to its original vial. The release vial was placed inside an oil bath on a heating plate (IKA® RCT basic). Release media must be filtered prior to entering the cuvette as solid particles would result in light scattering and, therefore, erratic measurements. For this a stainless steel filter was placed on the pipe entrance. Release experiments were conducted as follows: approximately 20 mg of cefuroxime-loaded SBA-15 powder or a cefuroxime-loaded monolith were placed in the glass vial. The filter and pipe exit were both placed inside the vial and set in place with laboratory film which also seals the vial to prevent evaporation of release media. After the setup is complete, 5 ml of release media were added using a syringe. A 1X phosphate buffered saline solution (PBS) was used as a release media, at 37ºC in order to simulate physiological conditions as closely as possible. Agitation was achieved due to the flow of release media, as using magnetic stirrers would crack monoliths. Time-based measurement specifications are as follows: the first measurement was conducted 15 s after the program was initialized (program should be initialized immediately after PBS is added to loaded mesoporous material), then every 60 s until 1000 s. After this, measurements were carried out with an increment of 10% in time with respect to the previous measurement given that release tends to be slower after the initial burst. Measurements are recorded for a maximum of 1,8·10 5 s (approximately 2 d). UV - Vis IV. RESULTS AND DISCUSSION 23 IV. Results and Discussion IV.1. MONOLITH AND MESOPOROUS MATERIALS CHARACTERIZATION Powder and monolithic SBA-15 were characterized using the techniques detailed in section III.3. The results obtained are explained in this section. IV.1.1. SEM Imaging The fibrous morphology of P-SBA-15 and M-SBA-15 can be clearly seen in SEM micrographs (Fig. IV.1). An image analysis to determine particle dimensions resulted in diameters between 4,5 and 7,5µm and lengths between 75 and 105µm, with a highly irregular distribution. The final micrograph shows how SBA-15 particles agglomerate to form the solid monolithic structure (see Fig. IV.2 for photographs of the final products). Fig. IV.1. SEM micrographs of synthesized fibrous SBA-15. The final micrograph is an image of a longitudinal section of a monolith. IV. RESULTS AND DISCUSSION 24 Fig. IV.2. Calcined monoliths. IV.1.2. TEM Imaging TEM micrographs (Fig. IV.3) of P-SBA-15 show well-defined pores and a uniform pore size distribution. An image analysis to determine pore size resulted in an average of 5,60±0,12 nm. The characteristic hexagonal pore structure of SBA-15 can be clearly seen on the inset micrograph. Fig. IV.3. TEM micrographs of SBA-15 powders. IV. RESULTS AND DISCUSSION 25 IV.1.3. SA-XRD Analysis SA-XRD spectra for P-SBA-15 (Fig. IV.4) show three clear peaks which can be indexed as (1 0), (1 1) and (2 0) reflections. These are characteristic of the reflections of a 2D hexagonal symmetry (p6mm). 0123456789 -5000 0 5000 10000 15000 20000 25000 30000 35000 40000 (2 0) (1 1) (1 0) Intensity 2θ P-SBA-15 Fig. IV.4. Small-angle X-Ray Diffraction spectrum for P-SBA-15. Three characteristics peaks of a hexagonallyordered structure are shown. IV.1.4. Nitrogen adsorption analysis Materials pore shape and size, specific surface area and specific pore volume were determined by nitrogen adsorption evaluations. Adsorption isotherms are functions that relate the amount of adsorbate adsorbed at equilibrium, to the pressure of the adsorbate in the gas phase, at a constant temperature. Adsorption of a gas depends, amongst other things, on the porous structure of the adsorbant. Therefore it is possible to determine additional information on pore shape by adsorption isotherm analysis. 0,0 0,5 1,0 0 300 600 900 Quantity Adsorbed (cm³/g STP) Relative Pressure (p/p°) Ads. P-SBA-15 Des. P-SBA-15 Ads. M-SBA-15 Des. M-SBA-15 Ads. M-SBA-15' Des. M-SBA-15' Fig. IV.5. Adsorption/desorption isotherms. IV. RESULTS AND DISCUSSION 26 According to the IUPAC, there are six types of physical adsorption isotherms, and four types of hysteresis43. In Fig. IV.5, the adsorption isotherms of P-SBA-15 and the monolithic forms are shown. The isotherms can be classified as type V, which are associated to mesoporous structures. Hysteresis in corresponds to the type H-1, which is characteristic of well-defined cylindrical pores and uniform pore size distribution. This information confirms the information collected from TEM micrographs in which an ordered and uniform mesoporous structure was observed. Using the BET theory model, the specific surface areas of each material were determined. Pore sizes and specific pore volume were calculated using the BJH theory. The table below summarizes these properties. Table IV.1. SBA-15 powder and monolith properties determined by N2 adsorption. P-SBA-15 M-SBA-15 M-SBA-15’ Pore shape Cylindrical Cylindrical Cylindrical Pore size (nm) 6,9 6,8 6,7 Specific surface area (m2/g) 914,6 547,0 294,4 Specific pore volume (cm3/g) 1,16 0,90 0,50 P-SBA-15: Powder SBA-15, M-SBA-15: Monolithic SBA-15; M-SBA-15’: APTES modified monolithic SBA-15 There is a reduction in specific surface area and pore volume after functionalization, due to the incorporation of organic moieties. It has been suggested that this reduction can be attributed to the blockage of much smaller pores (>2nm) that may be present with a broad distribution within the mesoporous materials44. The grafting of organic moieties modifies the silica surface, making it difficult for nitrogen molecules form the monolayer necessary for N2 adsorption measurements on the material surface. Upon applying the aforementioned mathematical models to determine specific surface area and pore volume, a reduction is obtained due to the direct dependency of the models to the amount of N2 adsorbed. As was detailed in sections III.1 and III.2, materials were functionalized with (3-Aminopropyl)thriethoxysilane (APTES). The approximate size of this molecule is 1,3nm in length and 0,7 nm in diamterer45. The reduction in specific surface area and pore volume between the powder and monolithic form of SBA-15 can be attributed to the incorporation of an additional Si layer on the surface of the material, aside from the pore blockage previously mentioned. As was explained in section III.2, monoliths are prepared with previously functionalized SBA-15 powders due to the fact that they showed a higher mechanical stability than those prepared with unfunctionalized SBA-15 (see section IV.1.6). Upon calcination during monolith preparation, the organic tails of APTES are lost, but the additional Si atom remains within the structure. Further functionalization (M-SBA-15’) would result in an even lower specific surface area and pore volume. The change in mesopore size however is hardly significant, suggesting that grafting of small organic moieties on larger pores does not affect their size, but again confirming that blockage of micropores with the same moieties is possible. IV. RESULTS AND DISCUSSION 27 IV.1.5. Mercury porosimetry analysis Further characterization of the porous structure of SBA-15 monoliths was carried out by mercury intrusion porosimetry evaluations. Due to the shape of SBA-15 particles, monoliths have a bimodal array of pores: the characteristic mesopores of SBA-15 and macropores in the range of microns that correspond to the interstitial sites formed within the monolith (see section IV.1.1). Fig. IV.6 shows the intrusion and extrusion curves of M-SBA-15 and M-SBA-15’. Pore sizes and interstitial and total porosity of monoliths are presented in the table below. 1000000 100000 10000 1000 100 10 1 0 1 2 3 4 5 Cumulative Intrusion (ml/g) Pore size Diameter (nm) Intrusion M-SBA-15 Extrusion M-SBA-15 Intrusion M-SBA-15' Extrusion M-SBA-15' Fig. IV.6. Hg porosimetry of unmodified (SBA-15) and modified (SBA-15+APTES) monoliths. Interstitial pores are larger than the mesopores and therefore hold more mercury than particle pores. Moreover, unfunctionalized monoliths will hold more mercury than their functionalized counterparts; total intrusion volume for M-SBA-15 was 4,756 ml/g, while for M-SBA-15’ intrusion reached 2,348 ml/g. Two pore sizes are discernible in Fig. IV.6 (intrusion curve), corresponding to the pore distribution described above. Table IV.2. SBA-15 monolith properties determined by Hg porosimetry. M-SBA-15 M-SBA-15’ Interstitial pore size (µm) 3,43 3,19 Interstitial porosity (%) 47,6 47,6 Total porosity (%) 84,5 83,4 Interstitial pores of M-SBA-15 are slightly larger than those of M-SBA-15’, and total porosity is reduced after functionalization, most likely due to micropore blockage as was described in the previous section. The slight decrease in total porosity is also due to pore blockage. IV. RESULTS AND DISCUSSION 28 IV.1.6. Thermogravimetric Analysis Organic modification of SBA-15 powders and monoliths was confirmed via TGA. The figure below show TGA and derivative TGA curves of modified and unmodified SBA-15 powders and monoliths. A peak is clearly discernible in the derivative curve for the temperature range of 400 to 600ºC46, which is attributed to the decomposition of APTES in a nitrogen atmosphere. This indicates effective surface modification. 0 400 800 84 90 96 Weight (%) Temperature (؛C) P-SBA-15 P-SBA-15' 0,00 0,02 0,04 Deriv. Weight (%/؛C) P-SBA-15 deriv. P-SBA-15' deriv. Fig. IV.7. TGA spectra for APTEs-modified SBA-15 powders. 0 200 400 600 800 1000 93 94 95 96 97 98 99 100 Weight (%) Temperature (؛C) M-SBA-15 M-SBA-15' 0,00 0,01 0,02 0,03 Deriv. Weight (%/؛C) M-SBA-15 deriv. M-SBA-15' deriv. Fig. IV.8. TGA spectra for APTES-modified SBA- monoliths. Monoliths showed a 3,2% in weight of effective surface modification versus 5,9% for powders. Monoliths yield a lower silanization given that they are prepared using previously functionalized SBA-15 powders. Although the organic groups of APTES are lost after monolith calcination, a decrease in the hydroxyl sites available for functionalization with respect to the IV. RESULTS AND DISCUSSION 29 powder counterpart is possible, resulting in a less effective organic modification. Moreover, diffusion of the functionalization agents to the interior of monoliths is difficult given their macroscopic morphology. IV.1.7. Mechanical stability assays Mechanical stability of monoliths was determined by calculating the difference in mass for monoliths left for 21 days in PBS at 37ºC. Mass loss for SBA-15 monoliths prepared with unfunctionalized material was of 37,4%, while for those prepared with functionalized SBA-15 mass loss was 4,2%. Monoliths prepared with functionalized SBA-15 proved to be more resistant to dissolution in aqueous media, for this reason they were chosen as matrices for drug delivery experiments. IV.2. CEFUROXIME RELEASE IV.2.1. Cefuroxime loading Materials under study (P-SBA-15, P-SBA-15’, M-SBA-15 and M-SBA-15’) were soaked during 24h in a solution of the model antibiotic (cefuroxime sodium salt, CEF) to entrap drug molecules within the available pores (see section III.4). Effective uptake of CEF was confirmed by TGA (see Fig. IV.9-Fig. IV.11). A peak is observed in the derivative curve from 100 to 110ºC which corresponds to elimination of water from samples. From 200 to 300ºC, a peak is discernible which can be attributed to decomposition of cefuroxime. As was previously mentioned, the peak in the range from 400 to 600ºC corresponds to the decomposition of APTES in the sample. 0 200 400 600 800 1000 20 30 40 50 60 70 80 90 100 110 Weight (%) Temperature (؛C) CEF 0,0 0,3 0,6 CEF deriv. Deriv. Weight. (%/؛C) Fig. IV.9. TGA spectra for cefuroxime sodium salt. VI. REFERENCES 37 VI. References 1 Rouquerol, J. et al. Recommendations for the characterization of porous solids. Pure & Appl. Chem. 66, 1739-1758, doi:10.1351/pac199466081739 (1994). 2 Chiola, V., Ritsko, J. E. & Vanderpool, C. D. Process for producing low-bulk density silica. (1971). 3 Di Renzo, F., Cambon, H. & Dutartre, R. A 28-year-old synthesis of micelle-templated mesoporous silica. Microporous Materials 10, 283-286 (1997). 4 Yanagisawa, T., Shimizu, T., Kuroda, K. & Kato, C. The Preparation of Alkyltriinethylaininonium– Kaneinite Complexes and Their Conversion to Microporous Materials. Bulletin of the chemical society of Japan 63, 988-992, doi:10.1246/bcsj.63.988 (1990). 5 Kresge, C. T. et al. M41S: A new family of mesoporous molecular sieves prepared with liquid crystal templates. Vol. 92 (Elsevier Science Publ B V, 1995). 6 Yang, P., Zhao, D., Margolese, D. I., Chmelka, B. F. & Stucky, G. D. Block Copolymer Templating Syntheses of Mesoporous Metal Oxides with Large Ordering Lengths and Semicrystalline Framework. Chemistry of Materials 11, 2813-2826, doi:10.1021/cm990185c (1999). 7 Zhao, D. et al. Triblock Copolymer Syntheses of Mesoporous Silica with Periodic 50 to 300 Angstrom Pores. Science 279, 548-552, doi:10.1126/science.279.5350.548 (1998). 8 Melde, B. J., Holland, B. T., Blanford, C. F. & Stein, A. Mesoporous Sieves with Unified Hybrid Inorganic/Organic Frameworks. Chemistry of Materials 11, 3302-3308, doi:10.1021/cm9903935 (1999). 9 Yang, P., Zhao, D., Margolese, D. I., Chmelka, B. F. & Stucky, G. D. Generalized syntheses of largepore mesoporous metal oxides with semicrystalline frameworks. Nature 396, 152-155 (1998). 10 Song, S. W., Hidajat, K. & Kawi, S. Functionalized SBA-15 Materials as Carriers for Controlled Drug Delivery: Influence of Surface Proper_es on Matrix−Drug Interac_ons. Langmuir 21, 9568-9575, doi:10.1021/la051167e (2005). 11 Trewyn, B. G., Giri, S., Slowing, I. I. & Lin, V. S. Y. Mesoporous silica nanoparticle based controlled release, drug delivery, and biosensor systems. Chemical Communications, 3236-3245 (2007). 12 Ma, Y. et al. Large-pore mesoporous silica spheres: synthesis and application in HPLC. Colloids and Surfaces A: Physicochemical and Engineering Aspects 229, 1-8, doi:10.1016/j.colsurfa.2003.08.010 (2003). 13 Ryoo, R., Joo, S. H., Kruk, M. & Jaroniec, M. Ordered Mesoporous Carbons. Advanced Materials 13, 677-681 (2001). 14 Schüth, F. Non-siliceous Mesostructured and Mesoporous Materials†. Chemistry of Materials 13, 3184-3195, doi:10.1021/cm011030j (2001). 15 Beck, J. S. et al. Molecular or Supramolecular Templating: Defining the Role of Surfactant Chemistry in the Formation of Microporous and Mesoporous Molecular Sieves. Chemistry of Materials 6, 1816-1821, doi:10.1021/cm00046a040 (1994). 16 Vinu, A., Mori, T. & Ariga, K. New families of mesoporous materials. Science and Technology of Advanced Materials 7, 753-771 (2006). VI. REFERENCES 38 17 Katiyar, A., Yadav, S., Smirniotis, P. G. & Pinto, N. G. Synthesis of ordered large pore SBA-15 spherical particles for adsorption of biomolecules. Journal of Chromatography A 1122, 13-20, doi:10.1016/j.chroma.2006.04.055 (2006). 18 Zhao, D. et al. Continuous Mesoporous Silica Films with Highly Ordered Large Pore Structures. Advanced Materials 10, 1380-1385, doi:10.1002/(sici)1521-4095(199811)10:16<1380::aidadma1380>3.0.co;2-8 (1998). 19 Schmolka, I. R. Polyoxyethylene-polyoxypropylene aqueous gels. (1966). 20 Raman, N. K., Anderson, M. T. & Brinker, C. J. Template-Based Approaches to the Preparation of Amorphous, Nanoporous Silicas. Chemistry of Materials 8, 1682-1701, doi:10.1021/cm960138+ (1996). 21 Nakanishi, K. Pore Structure Control of Silica Gels Based on Phase Separation. Journal of Porous Materials 4, 67-112, doi:10.1023/a:1009627216939 (1997). 22 Hoffmann, F., Cornelius, M., Morell, J. & Fröba, M. Silica-Based Mesoporous Organic–Inorganic Hybrid Materials. Angewandte Chemie International Edition 45, 3216-3251, doi:10.1002/anie.200503075 (2006). 23 Melosh, N. A. et al. Molecular and Mesoscopic Structures of Transparent Block Copolymer-Silica Monoliths. Macromolecules 32, 4332-4342 (1999). 24 Feng, P., Bu, X., Stucky, G. D. & Pine, D. J. Monolithic Mesoporous Silica Templated by Microemulsion Liquid Crystals. J. Am. Chem. Soc. 122, 994-995 (2000). 25 Melosh, N. A., Davidson, P. & Chmelka, B. F. Monolithic Mesophase Silica with Large Ordering Domains. Journal of the American Chemical Society 122, 823-829, doi:10.1021/ja992801b (2000). 26 Minakuchi, H., Nakanishi, K., Soga, N., Ishizuka, N. & Tanaka, N. Octadecylsilylated Porous Silica Rods as Separation Media for Reversed-Phase Liquid Chromatography. Analytical Chemistry 68, 3498-3501, doi:10.1021/ac960281m (1996). 27 Sun, Z. et al. Hierarchically Ordered Macro-/Mesoporous Silica Monolith: Tuning Macropore Entrance Size for Size-Selective Adsorption of Proteins. Chemistry of Materials 23, 2176-2184, doi:10.1021/cm103704s (2011). 28 Amatani, T., Nakanishi, K., Hirao, K. & Kodaira, T. Monolithic Periodic Mesoporous Silica with Well-Defined Macropores. Chemistry of Materials 17, 2114-2119, doi:10.1021/cm048091c (2005). 29 Leventis, N. et al. Polymer nano-encapsulation of templated mesoporous silica monoliths with improved mechanical properties. Journal of Non-Crystalline Solids 354, 632-644, doi:10.1016/j.jnoncrysol.2007.06.094 (2008). 30 Smått, J.-H., Schunk, S. & Lindén, M. Versatile Double-Templating Synthesis Route to Silica Monoliths Exhibiting a Multimodal Hierarchical Porosity. Chemistry of Materials 15, 2354-2361, doi:10.1021/cm0213422 (2003). 31 Liang, C., Dai, S. & Guiochon, G. Use of gel-casting to prepare HPLC monolithic silica columns with uniform mesopores and tunable macrochannels. Chemical Communications, 2680-2681 (2002). 32 Vasiliev, P. O., Shen, Z., Hodgkins, R. P. & Bergström, L. Meso/Macroporous, Mechanically Stable Silica Monoliths of Complex Shape by Controlled Fusion of Mesoporous Spherical Particles. Chemistry of Materials 18, 4933-4938, doi:10.1021/cm061205v (2006). VI. REFERENCES 39 33 Sachse, A. et al. Functional silica monoliths with hierarchical uniform porosity as continuous flow catalytic reactors. Microporous and Mesoporous Materials 140, 58-68, doi:10.1016/j.micromeso.2010.10.044 (2011). 34 Paolino, D., Fresta, M., Sinha, P. & Ferrari, M. in Encyclopedia of Medical Devices and Instrumentation (ed John G. Webster) 437-495 (John Wiley & Sons, Inc., 2006). 35 Costa, P. & Sousa Lobo, J. M. Modeling and comparison of dissolution profiles. European Journal of Pharmaceutical Sciences 13, 123-133, doi:10.1016/s0928-0987(01)00095-1 (2001). 36 Farokhzad, O. C. & Langer, R. Impact of Nanotechnology on Drug Delivery. ACS Nano 3, 16-20, doi:10.1021/nn900002m (2009). 37 Vallet-Regí, M., Balas, F. & Arcos, D. Mesoporous Materials for Drug Delivery. Angewandte Chemie International Edition 46, 7548-7558, doi:10.1002/anie.200604488 (2007). 38 Hudson, S. P., Padera, R. F., Langer, R. & Kohane, D. S. The biocompatibility of mesoporous silicates. Biomaterials 29, 4045-4055 (2008). 39 Izquierdo-Barba, I., Martinez, Á., Doadrio, A. L., Pérez-Pariente, J. & Vallet-Regí, M. Release evaluation of drugs from ordered three-dimensional silica structures. European Journal of Pharmaceutical Sciences 26, 365-373, doi:10.1016/j.ejps.2005.06.009 (2005). 40 Vallet-Regı, M., Doadrio, J. C., Doadrio, A. L., Izquierdo-Barba, I. & Pérez-Pariente, J. Hexagonal ordered mesoporous material as a matrix for the controlled release of amoxicillin. Solid State Ionics 172, 435-439, doi:10.1016/j.ssi.2004.04.036 (2004). 41 Doadrio, J. C. et al. Functionalization of mesoporous materials with long alkyl chains as a strategy for controlling drug delivery pattern. Journal of Materials Chemistry 16, 462-466 (2006). 42 Doadrio, A. L. et al. Mesoporous SBA-15 HPLC evaluation for controlled gentamicin drug delivery. Journal of Controlled Release 97, 125-132, doi:10.1016/j.jconrel.2004.03.005 (2004). 43 Sing, K. S. W. et al. Reporting Physisorption Data for Gas/Solid System with Special Reference to the Determination of Surface Area and Porosity. Pure & Appl. Chem. 57, 603-619 (1985). 44 Ryoo, R., Ko, C. H., Kruk, M., Antochshuk, V. & Jaroniec, M. Block-Copolymer-Templated Ordered Mesoporous Silica: Array of Uniform Mesopores or Mesopore−Micropore Network? The Journal of Physical Chemistry B 104, 11465-11471, doi:10.1021/jp002597a (2000). 45 ChemAxon. Chemicalize, <http://www.chemicalize.org/structure/#!mol=CCO%5BSi%5D%28CCCN%29%28OCC%29OCC&so urce=fp> (2012). 46 Qin, Z. et al. Influences of thermal pretreatment temperature and solvent on the organosilane modification of Al13-intercalated/Al-pillared montmorillonite. Applied Clay Science 50, 546-553 (2010).