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Development and Characterization of Lipid Nanoparticles prepared by Miniemulsion Technique Clara Patrícia Andrade Lopes Thesis to obtain the Master of Science Degree in Biotechnology Supervisor: Professor Luís Joaquim Pina da Fonseca Examination Committee Chairperson: Professor Duarte Miguel de França Teixeira dos Prazeres Supervisor: Professor Luís Joaquim Pina da Fonseca Member of the committee: Doctor Dragana Popovic Correia de Barros December 2014 Acknowledgments I would like to start by thanking my supervisor, Professor Luis Fonseca who accepted me and provides me all the means to developed this research thesis, sharing his knowledge with me and guiding me through this project with optimism and patience (even through the rough times and the dangerously close deadlines) and for always being present when needed. I would like to express my sincere gratitude to all BEBL members, professors and colleagues, for all opinions, questions, answers and suggestions to improve my project. To all my lab partners, for making me feel so welcome, giving me all the scientific and moral support and especially for contributing with the good environment which in my opinion is truly important because provides a greater motivation and commitment of the people. I would like to acknowledge all my friends (leirienses, escuteiros and fculianos) that in all my life, in one way or another, have helped and support me with their truly friendship. Finally, a deep acknowledgment goes to my family, my mother and sisters, who never stopped to support me through all my academic years, always encouraging my success and putting up with my bad mood and bad retorts on the endless hours of work. And last, but certainly not the least, to my father who taught me to work hard, ever smile and never give up! i Abstract Lipid nanoparticles are a promising alternative to traditional colloidal drug carriers. The main reason for this is related with the well-known conception that lipids promote oral drug absorption, because they undergo the same physiological mechanisms of food lipid digestion. Lipid nanoparticles also offer unique properties such as small size, large surface area, increased drug loading and stability (especially for lipophilic drugs), possibility of controlled drug release, no toxicity and high bioavailability. In the present work a medium chain fatty acid (MCFA) SLNs and NLCs based on MCFA and a natural oil were successfully prepared by sonication technique and employing a nonionic surfactant. The two anti-TB drugs, rifampicin and pyrazinamide, are entrapped into obtained lipid nanoparticles. The developed particles were characterized in terms of particle size, polydispersity index, zeta potential, morphology and encapsulation efficiency. Mean particle size of all formulations ranged between 69 ± 5 nm to 601 ± 100 nm, showing a suitable size for oral administration. The zeta potential obtained was negatively enough to ensure a good physical stability of the particles. Morphological studies by TEM showed spherical to oval SLNs and RIFNLCs with well-defined periphery. The rifampicin encapsulation efficiency range between 67 ± 7% and 85 ± 5%, while pyrazinamide encapsulation efficiency range between 14 ± 8% to 29±15%. In conclusion, despite being essential several further studies to ensure the efficacy of obtained lipid nanoparticles, the results from the present work pose a strong argument for lipid nanoparticles as a promising strategy for the oral delivery of anti-TB drugs. Keywords: Oral Administration; Solid Lipid Nanoparticles; Medium Chain Fatty Acid; Nonionic Surfactant; Tuberculosis ii Resumo As nanopartículas lipídicas são uma promissora alternativa aos sistemas coloidais de transporte de fármacos, absorção oral de fármacos, dado que sofrem os mesmos mecanismos fisiológicos da digestão dos lípidos provenientes dos alimentos. As nanopartículas lipídicas também oferecem propriedades únicas como tamanho reduzido, grande área de superfície, aumento da capacidade de carga, estabilidade e libertação controlada do agente activo dentro da partícula (especialmente para fármacos lipofílicos), aumento da biodisponibilidade e reduzida toxicidade. No presente trabalho, nanopartículas lipídicas sólidas (SLN) de um ácido gordo de cadeia média (MCFA) e vectores lipídicos nanoestruturados (NLC) baseados na combinação desse MCFA e óleo natural foram preparados com sucesso através da técnica de sonicação e recorrendo a um tensioactivo não iónico. Dois fármacos anti-TB, a rifampicina e a pirazinamida, foram internalizados dentro das partículas lipídicas. Estas foram caracterizadas em termos de dimensão, índice de polidispersão, potencial zeta, morfologia e eficiência de encapsulação. A dimensão média de todas as formulações variou entre 69 ± 5 nm e 601 ± 100 nm, exibindo dimensões apropriadas para administração oral. O potencial zeta obtido foi suficientemente negativo para garantir uma boa estabilidade física das partículas. Os estudos de morfologia feitos através de Microscopia Electrónica de Transmissão (TEM) mostram que tanto as SLNs como as RIF-NLC apresentam uma forma esférica ou oval e uma periferia bem definida. A eficiência de encapsulação da rifampicina variou entre 67 ± 7% e 85 ± 5%, enquanto a da pirazinamida variou entre 14 ± 8% e 29±15%. Concluindo, apesar de ser necessário realizar outros estudos, os resultados apresentados neste trabalho permitem prever que as nanopartículas lipídicas obtidas são uma alternativa promissora para o transporte de fármacos anti-TB por via oral. Palavras-chave: Administração Oral; Nanopartículas Lipídicas Sólidas; Ácidos Gordos de Cadeia Média; Tensioactivo Não-Iónico, Tuberculose iii Table of Contents Acknowledgments .......................................................................................................................... i Abstract ..........................................................................................................................................ii Resumo ......................................................................................................................................... iii Table of Contents ..........................................................................................................................iv List of Figures ................................................................................................................................vi List of Tables ................................................................................................................................ vii List of Abbreviations .................................................................................................................... viii Motivation and Aims of the Thesis ................................................................................................ix 1. Introduction ............................................................................................................................ 1 1.1. Lipid Nanoparticles ............................................................................................................ 2 1.1.1. Lipid Nanoparticles as Oral Drug Delivery System ....................................................... 5 1.1.1.1. 1.1.2. Toxicological Concerns ........................................................................................... 11 Preparation Techniques for Lipid Nanoparticles ......................................................... 13 1.1.2.1. High Pressure Homogenization Technique ............................................................. 13 1.1.2.1.1. Hot Homogenization Technique .......................................................................... 14 1.1.2.1.2. Cold Homogenization Technique ........................................................................ 14 1.1.2.2. Microemulsion Technique ....................................................................................... 15 1.1.2.3. Ultrasonication Technique ....................................................................................... 15 1.1.2.4. Solvent Emulsification Evaporation Technique ....................................................... 16 1.1.2.5. Double Emulsion Technique ................................................................................... 16 1.1.2.6. Solvent Emulsification Diffusion Technique ............................................................ 17 1.1.2.7. Solvent Injection Technique .................................................................................... 17 1.1.2.8. Phase Inversion Temperature Technique ............................................................... 18 1.1.2.9. Microchannel/Microfluidic Technique ...................................................................... 18 1.1.3. Characterization of Lipid Nanoparticles....................................................................... 19 1.1.3.1. 1.1.3.1.1. Measurement of Particle Size ................................................................................. 19 Dynamic Light Scattering..................................................................................... 19 1.1.3.2. Measurement of Zeta Potential ............................................................................... 20 1.1.3.3. Encapsulation Efficiency.......................................................................................... 22 1.1.3.4. Morphology .............................................................................................................. 22 1.1.3.4.1. 1.1.3.5. Lipid Crystallinity...................................................................................................... 23 Tuberculosis – Review .................................................................................................... 24 1.2. 2. Transmission Electron Microscopy ...................................................................... 23 Materials and Methods ........................................................................................................ 28 2.1. 2.1.1. Materials .......................................................................................................................... 28 Lipids ........................................................................................................................... 28 iv 2.1.2. Surfactant .................................................................................................................... 28 2.1.2.1. 2.1.3. Hexadecane ............................................................................................................ 29 Bioactive Compounds ................................................................................................. 29 2.1.3.1. Rifampicin ................................................................................................................ 29 2.1.3.2. Pyrazinamide ........................................................................................................... 30 2.1.3.3. β-carotene ............................................................................................................... 30 2.1.4. 2.2. Water ........................................................................................................................... 31 Methods ........................................................................................................................... 31 2.2.1. Preparation of Lipid Nanoparticles .............................................................................. 31 2.2.1.1. 2.2.2. Lyophilisation of Lipid Nanoparticles ....................................................................... 33 Characterization of Lipid Nanoparticles....................................................................... 33 2.2.2.1. Particle Size ............................................................................................................. 33 2.2.2.2. Zeta Potential .......................................................................................................... 33 2.2.2.3. Particle Morphology ................................................................................................. 34 2.2.2.4. Encapsulation Efficiency.......................................................................................... 34 3. Results and Discussion ....................................................................................................... 37 3.1. Study of Fabrication Parameters ..................................................................................... 37 3.1.1. Empty SLN .................................................................................................................. 37 3.1.1.1. Influence of Temperature ........................................................................................ 41 3.1.1.2. Influence of Lipid Content ........................................................................................ 41 3.1.1.3. Influence of Sonication ............................................................................................ 42 3.1.1.4. Lyophilisation ........................................................................................................... 43 3.1.1.5. Long-Term Stability ................................................................................................. 44 3.1.2. 3.2. Empty NLC .................................................................................................................. 46 Bioactive Compounds Loaded SLNs and NLCs ............................................................. 47 3.2.1. β-carotene ................................................................................................................... 48 3.2.2. Rifampicin .................................................................................................................... 49 3.2.3. Pyrazinamide ............................................................................................................... 50 4. Conclusions and Further Works .......................................................................................... 52 References .................................................................................................................................. 54 v List of Figures Figure 1 – Schematic structure of solid lipid nanoparticle (a) and nanostructured lipid carrier (b). ....................................................................................................................................................... 2 Figure 2 - Models of drug incorporation in Solid Lipid Nanoparticles. From [32]. ......................... 3 Figure 3 - Models of drug incorporation in Nanostructured Lipid Carriers. From [50]................... 4 Figure 4 - Various mechanisms of enhancement of drug bioavailability in the presence of lipids. ....................................................................................................................................................... 7 Figure 5 - Schematic diagram of lipid nanoparticles formation in the microchannel system. From [113] ............................................................................................................................................. 18 Figure 6 -Correlation differences between small and large particles. From [126]. ..................... 20 Figure 7 - Zeta-potential of a nanoparticle in solution. From [126]. ............................................ 21 Figure 8 – Schematic structure of M. tuberculosis cell wall and action site of first-line anti-TB drugs. Intercalation of hydrophilic arabinogalactan and hydrophobic mycolate containing layers creates an extremely impermeable envelope for antibiotic penetration. Small molecules and nutrients are transported through porin channels that are deposited through these layers. Adapted from [140]. ..................................................................................................................... 26 Figure 9- Structural formula of hexadecane ................................................................................ 29 Figure 10- Structural formula of rifampicin .................................................................................. 29 Figure 11 - Structural formula of pyrazinamide ........................................................................... 30 Figure 12 – Structural formula of β-carotene .............................................................................. 30 Figure 13 – Lipid nanoparticles production. Left: Lipid phase and aqueous phase in the warm water bath under same conditions (temperature and agitation). Top Right: Lipid phase (on the right) and aqueous phase (on the left) after weight. Middle Right: Addition of hot aqueous phase in melt lipid phase. Bottom Right: Sonication of hot pre-emulsion. ............................................. 32 Figure 14 – Pyrazinamide standard solutions used in constructing of the calibration curve, after reacting with alkaline sodium nitroprusside. Pyrazinamide concentration increases from left to right. ............................................................................................................................................. 35 Figure 15 - Rifampicin calibration curve ...................................................................................... 35 Figure 16 - β-carotene calibration curve ..................................................................................... 36 Figure 17 - Pyrazinamide calibration curve ................................................................................. 36 Figure 18- Z-ave and PDI of empty SLNs with different content of MCFA (SLN_1 represents the lowest lipid phase content and SLN_6 represents the highest lipid phase content), ranging from a lowest temperature (a) to highest temperature (d). .................................................................. 39 Figure 19 - Zeta Potential of empty SLNs with different content of MCFA (SLN_1 represents the lowest lipid phase content and SLN_6 represents the highest lipid phase content), ranging from a lowest temperature (a) to highest temperature (d). .................................................................. 40 vi Figure 20 - Macroscopic aspect of empty SLNs: (left) - non-sonicated sample; (right) – sonicated sample ........................................................................................................................ 42 Figure 21 – Powder resulting of SLN_1b_II lyophilisation .......................................................... 43 Figure 22 – Particle Size Distribution by Intensity of SLN_1b_II before and after lyophilisation. 44 Figure 23 - Particle Size Distribution by Intensity of SLN_4b_I after one day and 7 months after production. ................................................................................................................................... 45 Figure 24 – TEM image of SLN_4b_I after 7 months. ................................................................ 46 Figure 25 – Differences in macroscopic appearance of empty and loaded NLCs immediately before and after sonication. ......................................................................................................... 47 Figure 26- TEM image of β-carotene loaded NLC. ..................................................................... 48 Figure 27 - TEM image of rifampicin loaded NLC. ...................................................................... 50 Figure 28 - TEM image of pyrazinamide loaded NLC ................................................................. 51 List of Tables Table 1 – Examples of different Drug-Loaded Lipid Nanoparticles and their potential benefits for application in oral administration route. ......................................................................................... 8 Table 2 - Dosage forms of TB nanomedicine. From [149]. ......................................................... 27 Table 3 - Composition % (w/w) of bioactive compounds loaded to lipid nanoparticles. ............. 31 Table 4 - Physicochemical characteristics of empty SLN at different conditions (mean ± SD, n = 6). ................................................................................................................................................ 38 Table 5 – Physicochemical characteristics of the SLN_1b_II. Values are mean ± SD, n = 3 ..... 44 Table 6 - Physicochemical characteristics of the SLN_4b_I. Values are mean ± SD, n = 3. ..... 45 Table 7 – Physicochemical characteristics of the empty NLC. Values are mean ± SD, n = 3. ... 46 Table 8 - Physicochemical characteristics of the β-carotene lipid nanoparticles. Values are mean ± SD, n = 3. ....................................................................................................................... 48 Table 9 - Physicochemical characteristics of the rifampicin lipid nanoparticles. Values are mean ± SD, n = 3. ................................................................................................................................. 49 Table 10 - Physicochemical characteristics of the pyrazinamide lipid nanoparticles. Values are mean ± SD, n = 3. ....................................................................................................................... 51 vii List of Abbreviations ATM Atomic Force Microscopy RES Reticulo-Endothelial System CMC Critical Micellar Concentration RIF Rifampicin CNS Central Nervous System SD Standard Deviation DLS Dynamic Light Scattering SEM Scanning Electron Microscopy DSC Differential Scanning Calorimetry SLN Solid Lipid Nanoparticle EE Encapsulation Efficiency STM Scanning Tunnelling Microscopy ETB Ethambutol TB Tuberculosis FDA Food and Drug Administration TEM Transmission Electron Microscopy FFEM Freeze Fracture Electron Microscopy UV Ultraviolet GI Vis Visible GRAS Generally Recognized as Safe w/o Water-in Oil HHT Hot Homogenization Technique w/o/w Water-in Oil-in Water HLB Hydrophilic-Lipophilic Balance w/w Weight/Weight HPH High Pressure Homogenization WHO World Health Organization INH Isoniazid XDR Extremely Drug Resistant KatG Catalase Peroxidase Z-Ave Z-Average LC Loading Capacity ZP Gastrointestinal Zeta Potential MCFA Medium Chain Fatty Acid MDR Multi-Drug Resistant MTT (4,5-imethylthiazol-2-yl)2,5-dyphenyltetrazolium bromide NLC Nanostructured Lipid Carrier NP Nanoparticle o/w Oil-in Water OSPS Optical Single Particle Sizing PCS Photon Correlation Spectroscopy PDI Polydispersity Index PIT Phase Inversion Temperature PLG Poly(lactide-co-glycolide) PYZ Pyrazinamide R2 Determination Coefficient viii Motivation and Aims of the Thesis Tuberculosis (TB) remains one of major health problems in the world. In 2012, there were 8.7 million new cases of TB globally, with 1.4 million deaths in the same year. Treatment of TB is typically a 6-month regimen comprising an initial intensive phase of rifampicin (RIF), isoniazid (INH), pyrazinamide (PYZ), and ethambutol (ETB) daily for 2-months, followed by a 4-month continuation phase of RIF and INH given 3 times a week. The complexity of the treatment and patient non-compliance result in an incomplete or inadequate treatment and subsequently in the emergence of multi-drug-resistance. The epidemic of drug-resistant tuberculosis has spread quickly in some areas due to the convergence of resistant strains of M. tuberculosis in high-risk patients (e.g., those with human immunodeficiency virus/acquired immunodeficiency syndrome) and high-risk environments (e.g., hospitals and prisons). Moreover, the administration of antiTB drugs for a long period of time can lead to the incidence of unpleasant side effects, which decreases patient adherence and, therefore, increases costs of therapy. To overcome these drawbacks, new oral anti-TB formulations are required. Apart from some particular situations, the oral route is the first choice for drug administration. This preference is related with its easy access and non-invasive nature, which improves patient compliance and, therefore, facilitates treatments. However, the poor water solubility of several drug molecules and/or the risk of degradation throughout the gastrointestinal tract, turn into impossible their oral administration. In this perspective, efforts have been done in order to improve the oral bioavailability of poorly water-soluble drugs, by means of developing new colloidal delivery carriers. Among these systems are lipid nanoparticles, which have been showing promising results. The main reason for this is related with the well-known conception that lipids promote oral drug absorption, because they undergo the same physiological mechanisms of food lipid digestion. Lipid nanoparticles also offer unique properties such as small size, large surface area, increased drug loading and stability (especially for lipophilic drugs), possibility of controlled drug release, no toxicity and high bioavailability. Therefore, the first objective of the present work was to perform the production, characterization and study of a medium chain fatty acid (MCFA) SLNs and MCFA/natural oil NCLs prepared by sonication and employing a nonionic surfactant. The second objective of the project was to study the obtained SLNs and NCLs as an alternative system to improve the oral delivery of two anti-TB drugs, rifampicin and pyrazinamide. Furthermore, β-carotene was also used as lipophilic model. Its delivery has a great potential for food and cosmetic industry. ix 1. Introduction Nanotechnology continues to play a growing and tremendous interest, both on academic and industrial aspects. Such interest relies on the fact that it is now possible to manipulate nanometer-length atoms and molecules in order to create, according to a bottom-up technology, larger structures with outstanding properties. The conceptual underpinnings of nanotechnologies were first laid out in 1959 by the physicist Richard Feynman in his lecture, “There's plenty of room at the bottom” [1]. Feynman explored the possibility of manipulating material at the scale of individual atoms and molecules, imagining the whole of the Encyclopedia Britannica written on the head of a pin and foreseeing the increasing ability to examine and control matter at the nanoscale. However, the term 'nanotechnology' was not created until 1974, when Professor Norio Taniguchi of Tokyo Science University used it to refer to the ability to engineer materials precisely at the nanometer level [2]. In the last decade, drug delivery research is clearly moving from the micro to the nanometer scale. In simple terms, a drug delivery system is defined as a formulation or a device that enables the introduction of a therapeutic substance in the body and improves its efficacy and safety by controlling the rate, time, and place of release of this substance in the body. The process of drug delivery includes the administration of the therapeutic product, the release of the active ingredients by the product, and the subsequent transport of the active ingredients across the biological membranes to the site of action [3]. The new technologies employed in drug discovery lead to find many new powerful substances. However, the development of new drugs alone is not sufficient to ensure progress in drug therapy. Poor water solubility and insufficient bioavailability of the new drug molecules are main and common problems [4]. Therefore, there is an increasing need to develop a drug carrier system that overcomes these drawbacks. This carrier system should have no toxicity (acute and chronic), have a sufficient drug loading capacity and the possibility of drug targeting and controlled release characteristics. It should also provide chemical and physical stability for the incorporated drug. The feasibility of production scaling up with reasonable overall costs should also be required [5,6]. Colloidal drug delivery systems, particularly those in the nanosize range, have been increasingly investigated in the last years because they can fulfil the requirements mentioned above[7–9]. Size reduction is one of the methods to increase the solubility and hence the bioavailability of poorly-water soluble actives. Examples of such colloidal carriers are liposomes [10], nanoemulsions [11], micelles [12–15], polymeric nanoparticles[16–18], lipid nanoparticles [19,20], dendrimers [21–24] and drug nanocrystals [25–27]. Corresponding to the broad diversity of colloidal carriers, there are many possible administration routes: dermal [28–31], oral [32–36], parenteral [37,38], ocular [39], pulmonary [40–43] and intravenous [34,44]. According to the aim of this thesis, the work will only focus along of the text on lipid nanoparticles. The interested reader can find in the previous references high-quality reviews regarding the other carriers. 1 1.1. Lipid Nanoparticles Lipid nanoparticles were invented in the beginning of the nineties by two different researchers and co-workers, regarding their different production methods: the R.H. Müller and J.S. Lucks from Germany [45], and M.R. Gasco from Italy [46]. Nowadays, despite the number of academic groups interested in the study of these systems have been increased, R.H. Müller and co-workers are still the leaders of this research area [20]. In general, there are two types of lipid nanoparticles with a solid matrix, the solid lipid nanoparticles (SLNs) and the nanostructured lipid carriers (NLCs), which differ in their inner lipid structure (Figure 1). Figure 1 – Schematic structure of solid lipid nanoparticle (a) and nanostructured lipid carrier (b). SLNs consist of a solid lipid matrix that is solid at both room and body temperatures and that are prepared in a similar manner to an oil-in-water (o/w) emulsion, except that the oil phase of the emulsion is replaced by a solid lipid or a blend of solid lipids at room temperature [10]. Consequently, SLNs may be composed of a lipid or a mixture of lipids dispersed in an aqueous phase at high temperature and if necessary stabilized with a surfactant or combination of surfactants and then solidify at room temperature [10]. Particle size of SLNs range between 501000 nm [10]. The incorporation of drugs in SLNs can be described by three different models: the homogenous matrix model (in which drug is either molecularly dispersed or present as amorphous clusters in the lipid matrix); the drug-enriched shell model (outer lipid shell containing drug with lipid core) and the drug-enriched core model (drug core surrounded by lipid layer or reservoir type system) [10,32]. The three different models are depicted in Figure 2. 2 Figure 2 - Models of drug incorporation in Solid Lipid Nanoparticles. From [32]. The morphological differences between those models depend on the properties of the drug, on the matrix composition (in terms of lipids and surfactants), as well as on the method selected for their production. In many cases, a mixture of the three types of systems is obtained and, depending on the partition coefficient of the drug between the lipid and the aqueous phases, the drug molecules can also be adsorbed onto the surface of the systems being therefore not physicochemically protected by the matrix. The homogenous matrix model is assumed for entrapped drugs that can show prolonged release from SLNs. The drug-enriched shell model is obtained when phase separation occurs during the cooling process from the liquid oil droplet to the formation of SLNs. The lipid precipitates first, forming an almost drug-free lipid core. At the same time, the concentration of drug in the remaining liquid lipid increases continuously. Finally, the compound-enriched shell crystallizes. The drug-enriched core model is formed when the opposite mechanism as described for the former model occurs. In this case, the drug precipitates first and the lipid shell formed around this core will have distinctly less drug. This leads to a membrane-controlled release governed by Fick’s law of diffusion. This model is formed when the drug concentration is close to its saturation solubility in the melted lipid [10,32]. SLNs combine the advantages of early controlled drug delivery systems while minimise their shortcomings. In general, a solid core offers many advantages in comparison to a liquid core. Emulsions and liposomes usually show lack of protection of encapsulated drugs and drug release as a burst (emulsions) or noncontrolled (from liposomes). SLNs allow for a controlled release effect, whilst protect the drug against degradations, have good long term stability and higher drug loading capacity. Polymeric nanoparticles also possess a solid matrix identical to SLNs. However, SLNs can be manufactured using physiologically acceptable and biodegradable lipid materials that have a GRAS (Generally Recognized as Safe) status and require the use of lower amounts of organic solvents during production, which decreases the toxicity [28]. Finally, and by contrast with liposomes and polymeric nanoparticles, SLNs have low cost excipients and production techniques and are easily transferred to large scale. Nonetheless, certain drawbacks have been associated with the use of SLNs such as: SLNs dispersions contain high amount of water, drug loading capacity of SLNs are limited due 3 to crystalline structure of solid lipid, expulsion of encapsulated drug may take place during storage due to formation of a perfect crystalline lattice especially when SLNs are prepared from high purified lipid, drug release profile may change with storage time, polymorphic transitions are possible, particle growth is also possible during the storage, and gelation of the dispersion may take place during storage [47]. SLNs prepared from one highly purified lipid can crystallize in a perfect crystalline lattice that allows very small space for the incorporation of drugs (Figure 1a). Lipids crystallize in high energetic lipid modifications, α and β′, immediately after preparation of SLN. However, the lipid molecules undergo a time-dependent restructuring process leading to formation of the lowenergetic modifications, βi and β, during storage. Formation of this perfect lipid crystalline structure leads to expulsion of drug. Therefore, despite SLNs being interesting delivery systems, relatively low drug loading capacity and potential expulsion of the drug during storage led scientists to think about new strategies. Consequently, a second generation of lipid nanocarriers, referred to as nanostructured st lipid carriers (NLCs) were developed at the turn of the 21 century [48]. NLCs matrices consist of a less ordered lipid matrix with imperfections due to the mixtures of solid and liquid lipids (Figure 1b) [38]. There are three types of NLC: the imperfect type, the multiple type, and the amorphous type (Figure 3). The imperfect type is achieved by mixing solid lipids with small amounts of liquid lipids. If higher amounts of oil are mixed with the solid lipid, a different type of nanostructure is present. Here, the solubility of the oil molecules in the solid lipid is exceeded; this leads to phase separation and the formation of oily nanocompartments within the solid lipid matrix [48,49]. Many drugs show a higher solubility in oils than in solid lipids so that they can be dissolved in the oil and still be protected from degradation by the surrounding solid lipids. This type of NLC is called the multiple type, and can be regarded as an analogue to w/o/w emulsions since it is an oil-in-solid lipid-in-water dispersion. Since drug expulsion is caused by continuing crystallization or transformation of the solid lipid, this can be minimized by the formation of a third type, the amorphous type. Here, the particles are solid but crystallization upon cooling is avoided by mixing special lipids (e.g., hydroxyoctacosanylhydroxy-stearate and isopropylmyristate) [48,49]. Figure 3 - Models of drug incorporation in Nanostructured Lipid Carriers. From [50]. 4 In fact, the different NLC lead to the possibility of incorporating a high amount of drug inside these systems, and moreover, control of drug release can still be observed because NLC matrix is still solid. As a result, NLCs have several advantages such as: NLCs dispersions with higher solid content can be produced, drug loading capacity is better than SLNs, drug release profile can be easily modulated, drug leakage during storage is lower than SLNs, and production of final dosage forms (e.g., tablets, capsules) is possible. 1.1.1. Lipid Nanoparticles as Oral Drug Delivery System Oral route is the most preferred route for drug administration due to greater convenience, less pain, high patient compliance, reduced risk of cross-infection, and needle stick injuries. Major portion of the drug delivery market is occupied by oral drug delivery systems. However, oral drug delivery is continuously looking for new strategies due to factors such as low drug solubility, poor gastrointestinal (GI) absorption, rapid metabolism, high fluctuation in the drug plasma level, and variability due to food effects. In addition, the acidic environment and the presence of several enzymes in distinct parts of the GI tract increase the risk of occurrence drug degradation [51]. These factors may cause disappointing in vivo results leading to failure of the conventional delivery systems [52]. Intake of a high-fat meal leads to prolongation of GI tract residence time, stimulation of biliary and pancreatic secretions, stimulation of lymphatic transport, enhancement of intestinal wall permeability, reduction of metabolism and efflux activity, and alteration in mesenteric and liver blood flow, which significantly contribute to improve oral bioavailability of drugs [53,54]. Therefore, lipid-based delivery systems may reduce the inherent limitations of slow and incomplete dissolution of poorly soluble drugs and facilitate the formation of solubilised phase from which absorption may occur [55,56]. A normal healthy adult GI tract is able to daily hydrolyse about 100-140 g of dietary lipids (mainly in the form of triglycerides). Despite the exact body mechanisms for lipid processing remains unclear, the procedure can be divided in digestive, absorption and systemic blood uptake phases. Lipids digestion generally begins in the stomach where triglycerides are hydrolysed to diglycerides and fatty acids by the acid-stable lipases such as lingual lipase and gastric lipase. Acid lipases have a greater affinity for medium chain triglycerides when compared with long chain triglycerides and do not hydrolyse phospholipids or cholesterol esters. Acid lipases are also inhibited by long chain fatty acid digestion products, (which are mostly protonated at gastric pH), and therefore digestion via acid lipases accounts for only approximately 10 to 30% of the overall hydrolysis of ingested triglycerides in food. Afterwards, gastric contents reach the duodenum, the first section of small intestine, where the presence of lipids stimulates both productions of lipase/co-lipase enzymes by pancreas and bile salts (phospholipids and cholesterol) by the gall bladder. Bile salts adhere to the surface of emulsion droplets promoting the lipase/co-lipase action and originating free fatty acids and colloidal 5 species like micelles, mixed micelles, vesicles. Micelles are composed by surfactant molecules which self-assemble in aqueous solution above a determinate concentration, the so-called critical micellar concentration (CMC). Mixed micelles are similar to micelles but are composed of various surfactants molecules. Vesicles are formed by the self-assembling of insoluble phospholipids. Absorption occurs mostly in the small intestine where occurs the passage of substances directly to systemic circulation, or firstly to lymphatic circulation and subsequently to blood [57,58]. Concerning the typical lipid nanoparticles triglyceride-based composition, is expected that after oral administration, they undergo similar mechanisms of food-ingested lipids. The size of nanoparticle is an important factor for uptake into the epithelial of GI. Intestinal cells cannot absorb nanoparticles larger than 400 nm [59]. Furthermore, lipid nanoparticles have adhesive properties, which permit their adherence to the enterocytes (intestinal epithelial cells) surface. Therefore, the drug release from the nanoparticles is immediately followed by direct absorption within the enterocytes. In parallel, the presence of lipid nanoparticles in the duodenum promotes both lipase/co- lipase activities and bile salts secretion. The former hydrolyse the triglycerides in monoglycerides and fatty acids forming micelles, which undertake (i.e. re-solubilise) the drug meanwhile it is released during the degradation of the nanoparticles. Additionally, the bile salts interact with micelles and form mixed micelles. Subsequently, drug is absorbed together with these colloidal species, by one or more of the following transport mechanisms (Figure 4): transcellular, which is elected by small lipophilic molecules and involve the passive passage throughout the intestinal epithelial cells; paracellular implies the passive passage through the aqueous pores existing between enterocytes (tight junctions) and, therefore, is the selected path by hydrophilic drug molecules; active carrier mediated require the association of drug molecules with a specific transporter or carrier, which undertake the passage through enterocytes; receptor mediated comprise cell internalization of drug molecules by means of processes like endocytosis, phagocytosis and pinocytosis. After crossing intestinal epithelia, the most part of drug molecules pass directly to the hepatic portal vein, which carries them to the liver and afterwards to the systemic circulation. This process constitutes a problem when drugs undertake first-pass metabolism, since they can be inactivated before reach their local of action. Nonetheless, highly lipophilic drugs (log P > 5) tend to entry mostly by lymphatic circulation, when comparing to portal circulation, which avoids the risk of occurring liver first-pass metabolism. Hence, lipid can augment lymphatic uptake of several drugs, especially lipophilic drugs or large molecular weight macromolecules. Furthermore, lymphatic capillaries are significantly more permeable to nanoparticles than the blood capillaries. However, lymphatic absorption depends on the length of the fatty acid chains. Small- and medium-chain lipids across the enterocytes by diffusion and enter directly to systemic circulation, while long-chain lipids pass first to endoplasmic reticulum where they are reesterified into triglycerides and associated with lipoproteins. Following they pass to lymphatic circulation and finally enter into systemic circulation. 6 Figure 4 - Various mechanisms of enhancement of drug bioavailability in the presence of lipids. Oral administration of SLNs is possible as aqueous dispersion [60] or alternatively transformed into a traditional dosage forms such as tablets, pellets, capsules, or powders in sachets [58]. Since the stomach acidic environment and high ionic strength favour the particle aggregation, aqueous dispersions of lipid nanoparticles might not be suitable to be administered as dosage form. In addition, the presence of food will also have a high impact on their performance [5]. The packing of SLNs in a sachet for redispersion in water or juice prior to administration will allow an individual dosing by volume of the reconstituted SLNs. For the production of tablets, the aqueous SLNs dispersions can be used instead of a granulation fluid in the granulation process. Alternatively, SLNs can be transferred to a powder (by spray-drying or lyophilisation) and added to the tableting powder mixture. In both cases, it is beneficial to have a higher solid content to avoid the need of having to remove too much water. For cost reasons, spray drying might be the preferred method for transforming SLNs dispersions into powders, with the previous addition of a protectant [48]. For the production of pellets, the SLNs dispersion can be used as a wetting agent in the extrusion process. SLNs powders can also be used for the filling of hard gelatine capsules. Several drugs (hydrophobic and hydrophilic) have been incorporated in lipid nanoparticles for oral administration. Table 1 shows some of these drugs encapsulated in SLNs and NLCs examples and their potential benefits for application in oral delivery. 7 Table 1 – Examples of different Drug-Loaded Lipid Nanoparticles and their potential benefits for application in oral administration route. Drug Amphotericin B Apomorphine Lipid Relevant effects Nanoparticle SLN SLN Improvement of oral bioavailability; Drug protection; In vivo drug controlled release; Improvement of oral bioavailability; Successful targeted drug to the local of Reference [61] [62] therapeutic action (brain striatum); Camptothecin Candesartan cilexetil Carvedilol SLN SLN SLN Clozapine SLN Cryptotanshinone SLN Curcumin SLN Cyclosporine A SLN Digoxin SLN Body distribution; Sustained release and tissue targeting; Improvement of oral bioavailability; Enhancement of oral bioavailability; Lymphatic uptake; Bypass hepatic first-pass metabolism; Improvement of oral bioavailability; Tissue distribution; Enhancement of GI absorption; Improvement of oral bioavailability; Change in metabolism; Improvement of oral bioavailability Prolonged release; Low variation in bioavailability; Enhancement of GI absorption Improvement of oral bioavailability; [63] [64] [65] [34] [66] [67] [68] [69] 8 Edelfosine Etoposide SLN NLC High accumulation of drug in the local of therapeutic action (brain); In vitro antiproliferative effects on glioma cells; In vivo reduction of tumor growth; Improvement of oral bioavailability; In vitro antiproliferative effects on lung [70] [71] carcinoma cells; Fenofibrate Insulin Insulin Lopinavir Lovastatin Melatonin Methotrexate N3-O-toluylfluorouracil N3-O-toluylfluorouracil Nitrendipine SLN Lectinmodified SLN SLN SLN NLC SLN SLN Cationic SLN Anionic SLN SLN Improvement of oral bioavailability Protection of insulin degradation by enzyme; Improvement of oral bioavailability; Enhancement of GI absorption; Enhancement of GI absorption; Importance of delivery site; Improvement of oral bioavailability; Effective target of the drug to the local of therapeutic action (CNS); Prolong the blood circulation time of the drug; Enhancement of encapsulation efficiency; Improvement of oral bioavailability; Improvement of stability in GI environment; Sustained delivery Improvement of oral bioavailability; Lymphatic transport; Enhancement of GI absorption; Enhancement of GI absorption; Improvement of oral bioavailability; Improvement of intestinal transport; Improvement of oral bioavailability; Reduction of first-pass metabolism; [72] [33] [73] [74] [75] [76] [23] [77] [60] [78] 9 Norfloxacin Ofloxacin Otcadecylamine Paclitaxel Pentoxifylline Praziquantel Puerarin SLN SLN SLN SLN SLN SLN SLN Improvement of oral bioavailability; Prolong the plasma drug level; Improvement of oral bioavailability; In vitro drug controlled release; In vitro enhanced antibacterial activity; Enhancement of GI absorption; Prolonged release; Lymphatic transport; .Improvement of oral bioavailability; In vitro drug controlled release; In vivo safety properties; Improvement of oral bioavailability; Reduction of first-pass metabolism; Improvement of oral bioavailability; Improvement of bioavailability; Prolonged systemic circulation; Improvement of oral bioavailability; Increase of drug concentrations in tissues, [79] [80] [81] [82] [83] [84] [85] especially for the target organs (heart and brain); Quercetin SLN Repaglidine SLN Enhancement of GI absorption; Improvement of oral bioavailability; Improvement of oral bioavailability; In vivo safety properties; Prolonged release; In vitro safety properties; In vitro drug controlled release; Improvement of oral bioavailability; [86] [87,88] Rifampicin, Isoniazid, and SLN [89] Pyrazinamide Risperidone SLN Simvastatin SLN [36] [90] 10 Spironolactone SLN Improvement of oral bioavailability; [91] Tretinoin SLN In vitro drug controlled release; [92] Vinpocetine NLC Improvement of oral bioavailability; [93] Vinpocetine SLN Enhancement of GI absorption; Improvement of oral bioavailability; Enhancement of GI absorption; Improvement of oral bioavailability; Improvement of tissue uptake and distribution; Improvement of oral bioavailability. α-Asarone γ -Tocotrienol SLN SLN [94] [95] [96] 1.1.1.1. Toxicological Concerns Lipid nanoparticles are generally made of physiological and GRAS excipients and therefore, metabolic pathways exist decreasing the risk of acute and chronic toxicity. Their degradation is relatively fast in non-toxic compounds that are easily eliminated through physiologic metabolic pathways. The lipid matrix degradation occurs mostly by lipases whereas only a minor part is degraded by non-enzymatic hydrolytic processes [97]. Nonetheless, even for biodegradable nanoparticles, the use of high concentrations of the carriers can lead to toxicological concerns. Therefore, the fate of the carriers in the body should be clarified. The human immune system, by means of macrophages, recognize all external nanoparticles as hostile matter and quickly phagocyte and clearance them from the body. Nonetheless, these immunological specialized cells are present in limited areas of the body (e.g. lungs), which decrease the risks of interference with oral administered systems. Furthermore, the nanoparticles with sizes below to 100 nm can be internalized by all body cells [20,98]. Regarding the typical lipid nanoparticles sizes of 100-300 nm (size above 100 nm), which means that their cellular uptake is not expected and, therefore, no significant toxicological concerns exist [25]. 11 Toxicological studies should be first performed in vitro, using cell models that mimic the body conditions, in order to minimize the number of animal studies and to have an idea of the cytotoxicity of the systems in an early stage. Nonetheless, in vivo studies must always be performed before pass to human clinical trials, since in vitro studies sometimes use short time periods and small concentration ranges, which not allow for realistic conclusions about the cytotoxicity of the nanocarrier systems [99]. Silva et al. [36] studied the toxicity of SLN and risperidone loaded SLNs with Caco-2 cells by (4,5-dimthylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay. The Caco-2 cell line is a human colon epithelial cancer cell line often used as a model to mimic the GI tract conditions for toxicological and pharmacological studies [100]. MTT assay evaluates the mitochondrial function as a measurement of cell viability. Therefore, only live cells could react with the MTT reagent and the cell viability is detected in a very early stage. The results suggest that all formulations evaluated are biocompatible with Caco-2 cells and well tolerated by the GI tract. Similar results have been reported elsewhere [101,102]. The amount of viable cells after SLNs exposure was performed by the MTT assay with Caco-2 cell models. Other authors have also reported that SLN show biocompatibility, which increase their attractiveness for drugdelivery applications [101]. Several studies have reported that lipid nanoparticles systems are well tolerated and demonstrate low cytotoxicity, compared with other conventional nanocarriers [101,103]. Lipid carriers prepared with several lipids and emulsifying agents did not exhibit substantial cytotoxic effects in vitro. SLN prepared up to concentrations of 2.5% lipid do not exhibit any cytotoxic effects in vitro [104]. In fact, it has been shown that even concentrations higher than 10% of lipid led to a viability of 80% in cultures of human granulocytes [105]. In contrast, some polymeric nanoparticles showed complete cell death at concentrations of 0.5%. It can be assumed that the cytotoxicity of the SLNs can be mainly attributed to emulsifiers and preservative compounds which are used in the production of these systems. Additionally, depending on the route of administration, the toxicity requirements of the nanoparticles systems can change, being less for dermal, moderate for oral and limited for intravenous administration [20]. In conclusion, from the data obtained until now, the lipid nanoparticles formulations appear to fulfil the essential prerequisite to the clinical use of an oral colloidal carrier that means low cytotoxicity. 12 1.1.2. Preparation Techniques for Lipid Nanoparticles Since lipid nanoparticles dispersions at high temperatures are miniemulsions, miniemulsions preparation techniques can be applied or modified for lipid nanoparticles production. A miniemulsion, also referred as nanoemulsion or ultrafine emulsions, compose a particular class of emulsions consisting of colloidal dispersions with a droplet size between 20500 nm. The physical appearance of nanoemulsion is transparent or bluish for the smallest droplet sizes between 20–100 nm, or milky for sizes up to 500 nm [106]. Nanoemulsions are non-equilibrium systems and cannot be formed spontaneously. Nanoemulsion can be produced through two ways. There is either high-energy emulsification method or low-energy emulsification method. High-energy emulsification method includes high-pressure homogenization, sonication or high-shear mixing, whereas condensation method such as phase inversion temperature (PIT) is an example of low-energy emulsification method [107]. As several techniques have been developed for the production of lipid nanoparticles, resulting in different particles sizes and shapes, some parameter must be considered in order to choose the suitable preparation method. The size can influence the pharmacological properties of the particles, but it is not the unique parameter considered to compare the various techniques. Toxicological issues are also very important. The materials used must be biocompatible and biodegradable, while the use of solvents can be a relevant drawback, since they can remain in traces in the final product. From a technological point of view, the possibility to scale up the process is very important, but also the feasibility of the method is relevant. In fact, the use of expensive and complex machine can hamper the production on lab scale. Finally, the drug entrapment is very important. Nowadays more and more complex molecules are entrapped within lipid nanoparticles. These molecules have different physical and chemical properties (solubility, hydrophobicity, etc.) and stability issues (temperature, pH, etc.). The chosen preparation technique should be the most appropriate to enhance drug loading and encapsulation efficiency within the nanoparticles, without hampering the chemical stability of the molecule itself. The following sections describe different existing approaches for SLNs and NLCs formulations. However, in some instances combination of different methods has been utilized to prepare the lipid nanoparticles. 1.1.2.1. High Pressure Homogenization Technique Müller and Lucks were the first to prepare SLNs by applying high pressure homogenization (HPH) technique [108]. It makes use of high pressure homogenizer which is accessible from several manufacturers. In this technique, melted lipid solution is forced at a high pressure (100-2000 bar) through a narrow gap of few microns. The resulting high shear stress and cavitation forces decrease the particle size. A homogeneous dispersion with narrow size 13 distribution is desirable to increase the physical stability of the aqueous dispersion. If the particles localized at different positions in the dispersion volume, experience different forces then the degree of particle disruption will vary. There are two basic production methods to HPH: the hot and the cold homogenization techniques. In both the techniques, the lipid contents are in the range of 5-10%, but even higher concentration of lipid (40%) can be homogenized to nanodispersion [109]. 1.1.2.1.1. Hot Homogenization Technique In this method, the drug is first dissolved or solubilized in the lipid being melted at 5-10ºC above its melting point. The drug-containing melt is dispersed under stirring in a hot aqueous surfactant solution of identical temperature [110]. The pre-emulsion is then passed through a high pressure homogenizer (e.g. Micron LAB40) for 3 to 5 cycles and applying a pressure of about 500-1500 bar [111]. The obtained nanoemulsion is cooled down to room temperature or lower. The lipid recrystallizes and leads to SLNs. Homogenization pressure and the number of cycles should not be higher than that required to achieve the desired effects because this increases the cost of production and the chances of metal contamination as well as in some cases it would result in increase in particle size due to aggregation as a result of the high surface free energy of the particles [112]. This technique is performed at a high temperature and thus cannot be used for temperature sensitive drugs. So, to avoid heat-accelerated drug degradation, the length of time for drug exposure should be shortened [10]. This method is also not suitable for hydrophilic drugs because they would partition between the melted lipid and aqueous phase during hot homogenization [10]. Either medium scale or large scale production is possible for SLNs by HHT and it is the most extensively technique for the preparation of SLNs. 1.1.2.1.2. Cold Homogenization Technique In this process, the drug is also dissolved or solubilized in lipid being melted at 5-10ºC above its melting point. The mixture is rapidly cooled down with help of liquid nitrogen or dry ice. Rapid cooling procedure helps in the homogeneous distribution of the active ingredient. This solidified mixture is milled to about 50-100 μm particles using a ball or mortar mill and followed by dispersion of the lipid microparticles in a cold surfactant aqueous solution to obtain a suspension. This suspension is then passed through a high pressure homogenization at or below room temperature. Here, the cavitation forces are strong enough to break the lipid microparticles to SLNs. The cold homogenization technique reduces the chances of drug degradation induced by temperature and thus thermo label drugs can be used. Since the solid lipid is milled the complexity arising due to lipid modification can be avoided [112]. Chances of 14 drug distribution into the aqueous phase are limited and hence this method can be used for hydrophilic drugs as well as lipophilic drugs. Lipid nanoparticles prepared via this technique possess a slightly larger particle size and polydispersity when compared to the ones obtained by hot homogenization technique, using the same lipid at similar homogenization parameters (pressure, temperature and the number of cycles) but a higher number of homogenization cycles can be applied to reduce the particle size [10]. 1.1.2.2. Microemulsion Technique Gasco and co-workers were the first to develop SLNs based on the dilution of microemulsions [46]. Microemulsions are thermodynamically stable, clear and isotropic mixtures usually composed of an oil or lipid, emulsifier and/or co-emulsifier and water. In this process, the solid lipids are melted above their melting point. Separately, a mixture of emulsifier, coemulsifier and water is heated at same temperature. Both the lipid and the aqueous phase containing the emulsifier are mixed in appropriate ratios and stirred in order to produce a microemulsion. The hot microemulsion is then diluted with cold water (2-8°C) while stirring. The ratio of the hot microemulsion to cold water is usually in the range of 1:10 to 1:50 [113]. It has been noted that a droplet structure is already present in the microemulsion and therefore, no external energy is required to achieve the small particle size. When the microemulsion is diluted by cold water, the lipid droplets solidify as the temperature decreases. The temperature gradient and pH value determine the quality of the particles in addition to the composition of the microemulsion. Subsequently, the produced SLNs are washed three times with distilled water and followed by membrane filtration in order to remove any unwanted bigger lipid particle. Due to the dilution of microemulsion the concentrations of particle content are below 1% and, therefore, large amount of water has to be removed either by ultrafiltration or by lyophilisation in order to increase the particle concentration. The major limitation of this technique is its sensitivity to minor changes in composition or thermodynamic variables, which can lead to phase transitions. Lack of robustness of the microemulsion technique can lead to high production costs. Moreover, lipids solidification shifts the system to a thermodynamically unstable state. The high concentration of surfactants used may produce toxicity. Ultracentrifugation, ultrafiltration or dialysis can be applied to remove excess surfactants [113]. 1.1.2.3. Ultrasonication Technique In this technique, solid lipids are melted at 5–10°C above their melting points and drug is dissolved or dispersed in melted lipids. Then a hot aqueous surfactant solution (preheated at the same temperature) is added to the drug-lipid melt and homogeneously dispersed by a high shear mixing device. The coarse hot o/w emulsion obtained is ultrasonicated using a probe 15 sonicator till the desired sized nanoemulsion is formed. Finally, lipid nanoparticles are obtained by allowing hot nanoemulsion to cool down to room temperature. Two mechanisms are proposed for ultrasonic emulsification. First, the application of an acoustic field produces interfacial waves resulting in the dispersion of the oil phase in the continuous phase in the form of droplets [114]. Secondly, the application of ultrasound induces acoustic cavitation causing the formation and subsequent collapse of microbubbles by the pressure fluctuations of a simple sound wave, which creates extreme levels of highly localized turbulence. Therefore, the turbulent micro-implosions break up primary droplets into sub-micron size [115]. This technique is a simple and reproducible way to prepared nanoparticles without the need of organic solvents or any sophisticated instruments and has the potential to easily scale up for large scale production. However, metallic contamination of the product may occur during sonication by probe sonicator [116]. 1.1.2.4. Solvent Emulsification Evaporation Technique Sjöström and Bergenståhl were the first to describe the production of SLNs by solvent emulsification-evaporation technique [117]. The solid lipid is dissolved in a water immiscible organic solvent (e.g. cyclohexane, dichloromethane, toluene, chloroform, etc.) and the drug is dissolved or dispersed in the solution [118]. This organic phase containing the drug is emulsified in an aqueous surfactant solution by mechanical stirring. The organic solvent is then removed from the emulsion under mechanical stirring or reduced pressure (40-60 mbar) (e.g. rotary evaporator) [118]. Lipid nanoparticle dispersion is formed by the precipitation of the lipid phase in the aqueous surfactant medium. The mean particle size depends on the concentration of lipid in organic phase. Very small particle size can be obtained with low lipid load (5%) related to organic solvent. Aggregation of the particles can be avoided by removing the solvent at a faster rate [10]. Thermo labile drugs can be incorporated via this technique as it avoids thermal stress. Trace amounts of organic solvent remaining in the final product can potentially create toxicity problems. A large quantity of water has also to be removed during the final step of the formulation by means of ultrafiltration or evaporation [119,120]. 1.1.2.5. Double Emulsion Technique The double emulsion (w/o/w) method is based on solvent emulsification–evaporation method [113]. This method is mainly for the production of lipid nanoparticles loaded with hydrophilic drugs. In this case, drug is firstly dissolved in aqueous solvent (inner aqueous phase) and then is dispersed in lipid containing emulsifier/stabilizer to produce primary emulsion (w/o). A double emulsion (w/o/w) is formed after addition of an aqueous solution with a 16 hydrophilic emulsifier followed by stirring. Each emulsification step results in a highly polydisperse droplet distribution, exacerbating the polydispersity of the final double emulsion. Thus, lipid nanoparticles formed from double emulsions technique are, by nature, poorly controlled in both size and structure. 1.1.2.6. Solvent Emulsification Diffusion Technique Quintanar-Guerrero et al. were the first to describe this technique for the preparation of polymeric nanoparticles [121]. Recently this technique has been modified by various research groups for the preparation of lipid nanoparticles [122,123]. In solvent emulsification diffusion technique, the lipid is dissolved in a partially water miscible solvent (e.g. benzyl alcohol, isobutyric acid, or tetrahydrofuran) which is previously saturated with water at room temperature or at a controlled temperature in order to ensure the initial thermodynamic equilibrium [113]. The mixture is then emulsified in an aqueous solution of a surfactant by mechanical stirring at the temperature used to dissolve the lipid producing an o/w emulsion. This o/w emulsion is then diluted with excess water, in typical ratio ranges from 1:5 to 1:10 [113], at a controlled temperature which causes the diffusion of the solvent into the external phase and subsequent precipitation of the lipid nanoparticles. The diffused solvent can be removed either by vacuum distillation or ultrafiltration. The concentration and the nature of the lipid and surfactant, stirring rate and the processing temperature are critical variables in this technique. This technique avoids the necessity to melt the lipid which is very useful for preparation of protein and peptideloaded lipid nanoparticles. 1.1.2.7. Solvent Injection Technique The basic principle of the solvent injection method is similar to the solvent emulsification diffusion technique. In case of solvent injection method, lipids are dissolved in a water-miscible solvent (e.g., acetone, isopropanol, and methanol) or water-miscible solvent mixture and quickly injected into an aqueous solution of surfactants through an injection needle [113]. The advantages of this method are the easy handling and fast production process without technically sophisticated equipment (e.g., high-pressure homogenizer). However, the main disadvantage is the use of organic solvents. The resulted dispersion is filtered through a filter paper in order to remove any excess lipid [113]. The presence of emulsifier within the aqueous phase helps to produce lipid droplets at the site of injection and stabilize lipid nanoparticles until solvent diffusion was complete by reducing the surface tension between water and solvent. 17 1.1.2.8. Phase Inversion Temperature Technique The phase inversion temperature (PIT) method is commonly used for the preparation of nanoemulsions. The PIT concept uses the specific ability of some polyethoxylated surfactants to modify their affinities for water and oil as a function of the temperature. In the PIT nanoemulsion preparation method, the use of such surfactant type leads to an emulsion inversion from o/w macroemulsion to a w/o emulsion when temperature is increased above the PIT, and to the formation of an o/w nanoemulsion when the temperature is next lowered below the PIT [124]. Recently it has been adapted for the preparation of SLN. In this case two main components are used: an oil phase, constituted by solid lipids and non-ionic surfactants and an aqueous phase containing NaCl. The aqueous phase and the oil phase are separately heated above the PIT; then the aqueous phase is added dropwise, at constant temperature and under agitation, to the oil phase, in order to obtain a w/o emulsion. The mixture is then cooled to room temperature under slow and continuous stirring. At the PIT, the turbid mixture becomes clear, then below the PIT an o/w nanoemulsion is formed, which turns in lipid nanoparticles below the lipid melting point [125]. 1.1.2.9. Microchannel/Microfluidic Technique A novel microchannel system with a cross-shaped junction was developed by a main microchannel and two branches as shown in the Figure 5. The lipid dissolved in a watermiscible organic solvent is passed through the main channel, while simultaneously an aqueous surfactant solution is introduced into the branches. These two liquids met together at the crossshaped junction and passed along the main channel. The solvent diffused from the lipid solution stream into the aqueous phase, which resulted in the local supersaturation of lipid and thus led to the formation of lipid nanoparticles. The size of lipid nanoparticles prepared in the microchannel system was influenced either by velocities of the lipid and aqueous phases and by lipid and surfactant concentrations [113]. This technique allows precise control of the outer and inner drop sizes as well as the number of droplets encapsulated in each larger drop [113]. However, in a continuous production process, lipid nanoparticles contained in the liquid may block or deposit inside the microchannel, which could result in the break down and failure of particle preparation [113]. Figure 5 - Schematic diagram of lipid nanoparticles formation in the microchannel system. From [113] 18 1.1.3. Characterization of Lipid Nanoparticles Characterization of lipid nanoparticles is a challenge due to the small size of these colloidal carriers and complexity of the system. Numerous parameters need to be considered, such as mean particle size, zeta potential, drug association efficiency, degree of lipid crystallinity and lipid modification. For each parameter can be applied several techniques. However, only methodologies selected in this work will be further described. 1.1.3.1. Measurement of Particle Size Particle size might be determined by dynamic light scattering (DLS), optical single particle sizing (OSPS), laser diffraction (LD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), scanning tunnelling microscopy (STM) and freeze fracture electron microscopy (FFEM) [112,113]. 1.1.3.1.1. Dynamic Light Scattering Dynamic Light Scattering (DLS) also referred as Photo Correlation Spectroscopy (PCS) is a technique used to determine the mean particle size and the width of the particle size distribution expressed as polydispersity index (PDI). The particle size is the diameter of the sphere that diffuses at the same speed as the particle being measured. The measurement using DLS is based on the light scattering phenomena in which the statistical intensity fluctuations of the scattered light from the particles in the measuring cell are measured. These fluctuations are due to the random movement of the particles in the dispersion medium. This movement is called Brownian motion. Usually a DLS device consists of a laser light which illuminates a small volume of the sample composed by a dilute suspension of particles. The light scattered from these particles is collected by a lens and its intensity is measured by a photomultiplier at a certain angle (90º or 173º). The diffusion rate of the particles depends on their size (at a known fluid viscosity and temperature). Hence, the size of these particles can be calculated from the rate of fluctuation of the scattered light intensity. When the suspended particles are small, they diffuse relatively fast, and the fluctuations in the scattered light are rapid. On the other hand, if the particles are large, they move slowly, and the fluctuations in the scattered light are slow. The detected intensity signals are used by a correlator to calculate the auto-correlation function G(τ). A correlator is basically a signal comparator designed to measure the degree of similarity between two signals, or one signal with itself at varying time intervals. From the decay of correlation function, the diffusion coefficient (D) of the particles is obtained. Once the diffusion coefficient is known, the 19 hydrodynamic diameter of a spherical particle can be calculated applying the Stokes-Einstein equation (Eq.1), d(H) = 𝐾𝑇 3πηD Eq.1 where, d(H) is the hydrodynamic diameter, D is translational diffusion coefficient which measures the velocity of the Brownian motion, k is the Boltzmann’s constant, T is the absolute temperature and η is the viscosity of the solution. As it was previously mentioned, small particles diffuse faster than large ones, causing a stronger fluctuation in the scattering signal and a more rapid decaying G(τ) (Figure 6). For a monodisperse particle population, G(τ) is a single exponential, but if more than one size of particles is present the function is polyexponential. Deviation from a single exponential is used to calculate the PDI, which is a measure of the width of the size distribution. The PDI value is zero when a monodisperse particles population is measured. PDI values of around 0.10-0.30 indicate a relatively narrow distribution, values of 0.5 and higher are obtained in case of very broad distributions [126]. Figure 6 -Correlation differences between small and large particles. From [126]. 1.1.3.2. Measurement of Zeta Potential Almost all particles in contact with a liquid acquire an electric charge on their surface. The development of a nett charge at the particle surface affects the distribution of ions in the surrounding interfacial region, resulting in an increased concentration of counter ions close to the surface. Thus an electrical double layer exists around each particle (Figure 7). The liquid 20 layer surrounding the particle has an inner region called the Stern layer, where the ions are strongly bound and an outer and diffuse region, where the ions are less firmly attached. Within the diffuse layer there is an imaginary boundary inside which the ions and particles form a stable entity. When a particle moves, ions within the boundary move with it, but any ions beyond the boundary do not move with the particle. This boundary is called the surface of hydrodynamic shear or slipping plane. Zeta potential (ζ) is the potential that exists at this boundary and it is a parameter which is very useful for the assessment of the physical stability of colloidal dispersions [126]. Figure 7 - Zeta-potential of a nanoparticle in solution. From [126]. Charged particles suspended in the electrolyte are attracted towards the electrode of opposite charge, when an electric field is applied. Viscous forces acting on the particles tend to oppose this movement. When equilibrium between these two opposing forces is achieved, the particles move with a constant velocity. This velocity of the particle per unit electric field strength (E) is called the electrophoretic mobility (μ), which is expressed in micrometers per second per volt per centimeter (μm/s)/(V/cm). Zeta Potential can be measured by electrophoretic mobility through Henry’s equation (Eq. 2), μ= 2εζf(ka) 3η Eq. 2 where η is the viscosity of the dispersion medium, ε the permittivity of the environment (the dielectric constant) and f(ka) is the Henry’s function. Electrophoretic determinations of zeta 21 potential are most commonly made in aqueous media and moderate electrolyte concentration. In this case, f(Ka) is 1.5 and is referred to as the Smoluchowski approximation. Non-aqueous meassurements generally use the Huckel approximation and f(Ka) is 1 [126]. The storage stability of a colloid system can be predicted by the measurement of the zeta potential. It is currently accepted that absolute zeta potentials values higher than 30 mV are required for full electrostatic stabilization; potentials between 5 and 15 mV are in the region of limited flocculation; and lower than 3 mV of maximum flocculation. As a result, particle aggregation is less expected to occur for charged particles (high zeta potential) due to electric repulsion [113]. Nanoparticles with a zeta potential between -10 and +10 mV are considered approximately neutral, while nanoparticles with zeta potentials of greater than +30 mV or less than -30 mV are considered strongly cationic and strongly anionic, respectively. Since most cellular membranes are negatively charged, zeta potential can affect a nanoparticle's tendency to permeate membranes, with cationic particles generally displaying more toxicity associated with cell wall disruption. Positive charge possesses a membrane destabilizing and concomitantly destructive effect resulting from an interaction of positive charge and negative charge of membrane. Thus, positively charged lipids are not approved by Food and Drug Administration (FDA) for clinical use [127]. 1.1.3.3. Encapsulation Efficiency According to literature, the association efficiency of a drug in SLNs is most frequently assessed indirectly, meaning that the aqueous phase is separated from the lipid particles and subsequently drug content of the aqueous phase is determined [113]. The separation of the SLNs from the aqueous phase can be done by e.g. ultrafiltration, ultracentrifugation and gel separation. The amount of drug measured in the aqueous phase is considered the amount of drug not incorporated. The following equation (Eq. 3) is used to calculate the association efficiency: EE% = 𝑇𝐷−𝐹𝐷 TD × 100 Eq.3 where TD is total drug weighted at the beginning and FD is the free drug dissolved in aqueous medium. 1.1.3.4. Morphology Images of lipid nanoparticles could be achieved through several microscopic techniques, such as the scanning electron microscopy (SEM), transmission electron microscopy (TEM), 22 atomic force microscopy (AFM), CryoSEM and CryoTEM [112,113]. These techniques are very useful since allow the visualization and measurement of particles much smaller than 1 μm. 1.1.3.4.1. Transmission Electron Microscopy TEM utilizes energetic electrons to provide morphologic, compositional and crystallographic information of samples. At a maximum potential magnification of 1 nm, TEM is the most powerful microscope, producing high-resolution and two-dimensional images, allowing the investigation of micro- and nanostructures. Going through the several parts of the TEM system, first an electron gun generates a high energy electron beam by accelerating electrons emitted from a cathode. This acceleration voltage, usually greater than 100 kV, determines the microscope’s resolution. After the accelerator, there are condenser lenses to control beam diameter and convergence angles of the incident beam on a specimen. The TEM has three lenses to ensure good magnification capability, and the intermediate lens is used to switch the TEM between an image mode and a diffraction mode. TEM specimens must be thin foils (about 100 nm) because they should be able to transmit electrons, and this thin specimen is mounted in a 3 mm diameter grid. The specimen preparation depends on the type of sample. 1.1.3.5. Lipid Crystallinity Lipid crystallinity and polymorphic changes during storage are strongly related with drug incorporation and release rates [112,113]. Due to the small size of the SLN and the presence of surfactants, lipid crystallization and modification changes can be highly retarded. Consequently, special attention should be paid on the characterisation of such parameters. Lipid crystallinity and polymorphic forms are mainly investigated by differential scanning calorimetry (DSC) and X-ray scattering techniques [112,113]. DSC is a thermal analytical technique commonly used to characterize the physical state, the degree of crystallinity, polymorphism, crystal ordering, eutectic mixtures or glass transition processes in SLNs dispersions [112,113]. Additionally, DSC experiments are useful to understand drug and lipid interactions within SLNs. 23 1.2. Tuberculosis – Review Tuberculosis (TB) is an infectious disease caused by bacilli belonging to the Mycobacterium tuberculosis complex [128] and this complex consists of seven species including Mycobacterium tuberculosis, Mycobacterium canetti, Mycobacterium africanum, Mycobacterium pinnipedii, Mycobacterium microti, Mycobacterium caprae and Mycobacterium bovis [129]. TB is predominantly a disease of the lung, with pulmonary TB accounting for about 70% of the cases. Extra-pulmonary disease sites include lymph nodes, bone, and meninges [130] . The bacilli remain viable in the air for a long time and, eventually, they are inhaled during breathing, leading the bacteria directly to the lung where they are phagocyted by alveolar macrophages (white blood cells). Inside the macrophages, the bacilli reside in a membranebound vacuole, and for this reason some are able to avoid fusion with lysosomes and posterior digestion [131], ending up co-existing with the macrophages [132]. They multiply and eventually escape the lung through the bloodstream and lymphatic system, spreading to other organs of the body, resulting in the extra-pulmonary TB [133]. Moreover, M. tuberculosis may exist within a granulomas consisting of macrophages and giant cells, T cells, B cells, and fibroblasts, and these granulomas can prevail not only in the lung, but in other organs as well. In latent infections, the state of the bacteria within the granuloma is unknown. Despite 100 years of research, infection with M. tuberculosis is still a major health problem worldwide and one of the main causes of human death by a particular infectious agent. Inappropriate monotherapy and intermittent treatment are the main causes of drug resistance, and facilitate the selection and transmission of resistant strains within communities. In 2012, there were 8.7 million new cases of TB globally, with 1.4 million deaths in the same year, which confirms TB as a serious global health issue [128]. Of the 8.7 million incident TB cases in 2012, 1.1 million (13%) were among people living with HIV [128]. Currently available chemotherapy includes first-line drugs such as isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and pyrazinamide (PYZ) [134]. These drugs are administered orally and are shown to have excellent potency against M. tuberculosis. Isoniazid, a prodrug activated by the catalase peroxidase (KatG) enzyme, is the most well-known and used drug to treat TB [135]. Activated isoniazid inhibits NADH-dependent enoyl-ACP reductase, which plays a role in the synthesis and elongation of mycolic acids on the cell wall [135]. Rifampicin binds to the β-subunit of the RNA polymerase, preventing the formation of the messenger RNA chain [136]. Pyrazinamide is a prodrug converted into pyrazinoic acid by the pyrazinamidase enzyme [135]. The protonated pyrazinoic acid accumulates in the cell and causes cytoplasmic acidification and reduces cell membrane energy, disrupting the proton motive force and affecting membrane transport [136]. Ethambutol is a bacteriostatic agent that inhibits the polymerization of arabinogalactan, consequently preventing its biogenesis formation on the cell wall [137]. 24 The second-line drugs used for the treatment of TB are aminoglycosides (e.g. amikacin, kanamycin), polypeptides (e.g. capreomycin, viomycin, enviomycin), fluroquinolones (e.g. ciprofloxacin, levofloxacin, moxifloxacin), thioamides (e.g. ethionamide, prothioamide, cycloserine) [134]. They are used as alternative agents to treat TB when the bacilli are resistant to first-line drugs and they are less effective, more toxic, and unavailable in many countries due to high costs [138]. The effect of anti-TB drugs resistance has been around since the first anti-TB agent, streptomycin, appeared on the market as a monotherapy. Multi-drug resistant (MDR) TB is defined by resistance to, at least, rifampicin and isoniazid, whereas extensively drug-resistant (XDR) TB is defined as MDR-TB with additional resistance to any fluoroquinolone and to, at least, one of the injectable drugs (capreomycin, kanamycin and amikacin). Drug resistance in TB is the result of spontaneous mutation in combination with selection by poor programmatic and individual care performance [139]. Globally, WHO estimates that 3.7% of new tuberculosis cases and 20% of re-treatment cases have MDR-TB [128]. By the end of 2012, 84 countries had reported at least one case of extensively drug resistant strains (XDR-TB) [128]. M. tuberculosis is intrinsically resistant to numerous antibiotics, and only a few drugs are effective for treatment [140]. This intrinsic resistance is partially due to the thick, lipid-rich cell wall, which is an important characteristic of mycobacteria, limiting the penetration of antibiotics. The cell wall consists of a layer of peptidoglycan surrounding the cell’s basic lipid bilayer. A second layer of arabinogalactin runs parallel to the peptidoglycan layer, surrounding it in an intricate, sugary shell. A third layer extends perpendicular to the arabinogalactin shell and consists of a complex network of mycolic acids. These long and “sticky” mycolic acids are tightly bound in a final exterior layer, rendering the bacilli virtually impermeable and almost entirely waterproof. This complex armor allows M. tuberculosis to be resistant to many antibiotics, avoid death by acidic and alkaline compounds, and prevent cellular phagolysosomal fusion, allowing it to successfully evade lysis. Figure 8 depicts a simple visual representation of M. tuberculosis cell wall. Moreover, mycobacteria possess various defence systems against the activity of antibiotics, including potent β-lactamases and other drug-neutralizing enzymes [140]. Unlike many other bacteria, M. tuberculosis harbors no resistance plasmids, and drug resistance arises through the acquisition of specific chromosomal mutations [141]. The current short-course treatment guideline aims for a complete elimination of active and dormant bacilli involves two phases. During the initial phase four drugs (usually isoniazid, rifampicin, pyrazinamide and ethambutol) are given daily for two months. The continuation phase in which fewer drugs (usually isoniazid and rifampicin) are administered for an additional 4 months, either daily or 3 times per week, targets the killing of any remaining or dormant bacilli in order to prevent relapse [128]. 25 Figure 8 – Schematic structure of M. tuberculosis cell wall and action site of first-line anti-TB drugs. Intercalation of hydrophilic arabinogalactan and hydrophobic mycolate containing layers creates an extremely impermeable envelope for antibiotic penetration. Small molecules and nutrients are transported through porin channels that are deposited through these layers. Adapted from [140]. The search for new anti-TB drugs is an important key in this fight, but searching for new drug delivery strategies may also play an important role. Alternative delivery systems, such as nanocarriers for anti-TB drugs, may reduce administration frequency and shorten periods of treatment, hence improving patient compliance and efficacy of treatment, and reduce drug related toxicity. For example, isoniazid, rifampicin and pyrazinamide are limited by hepatotoxicity [142], a side effect due to the action of the drug on hepatocytes rather than macrophages, the primary host cells that harbor M. tuberculosis. Thus, a delivery mechanism that introduces these antibiotics selectively into macrophages would greatly increase their therapeutic efficacy by achieving higher concentrations of the antibiotics in site of infection without exposing the patient to high systemic concentrations that cause toxicities. Because M. tuberculosis resides and multiplies within host mononuclear phagocytes and because mononuclear phagocytes internalize particles more efficiently than other host cells, encapsulation of anti-TB drugs in nanoparticles offers a mechanism for specific targeting of M. tuberculosis-infected cells. Indeed, because nanoparticles have been shown to be taken up by macrophages of the reticuloendothelial system and to accumulate in the liver, spleen and lung [143–145], they are ideally suited to treat M. tuberculosis, which infects macrophages in these organs. As macrophages exhibit a number of receptors, it is also possible to modify the surface of these nanocarriers to achieve active targeting of macrophages. Sugars, such as mannose 26 [146] and lactose [147], are among the most commonly used for this purpose, since these receptors are highly expressed in macrophages. Particle size is also an important characteristic in passive targeting of macrophages, since they affect the success of internalization within these cells. In this regard, particles with diameters of about 500 nm have been reported as ideal to undergo phagocytosis by alveolar macrophages [148]. A variety of different nanocarriers have been produced and tested in vitro and in vivo as delivery platforms for anti-TB drugs. Table 2 summarizes major data on anti-TB nanoparticulate formulations. Table 2 - Dosage forms of TB nanomedicine. From [149]. Sustained release Mode of delivery and dosage forms Drug evaluated (days) Animal model Plasma Organs Oral PLG NPs RIF, INH, PZA Mice, guinea pigs 6−8 9−11 EMB Mice 3 7 Streptomycin Mice 4 7 Ethionamide Mice 6 5−7 Econazole Mice 5 6 Moxifloxacin Mice 4 6 Lectin-PLG NPs RIF, INH, PZA Guinea pigs 6−14 15 Alginate NPs RIF, INH, PZA, EMB Mice, guinea pigs 7−11 15 Econazole Mice 6 8 RIF, INH, PZA Mice 8 10 PLG NPs, nebulized RIF, INH, PZA Guinea pigs 6−8 9−11 PLG NPs, insufflated RIF Guinea pigs 0.25 0.3 Lectin-PLG NPs RIF, INH, PZA Guinea pigs 6−14 15 Alginate NPs RIF, INH, PZA, EMB Guinea pigs 7−11 15 SLNs RIF, INH, PZA Guinea pigs 5 7 32 36 SLNs Inhalable Injectable PLG NPs RIF, INH, PZA Mice 27 2. Materials and Methods 2.1. Materials 2.1.1. Lipids All lipids used in this work are well tolerated for human use and recognized as safe (GRAS status) by FDA. 2.1.2. Surfactant In this work was used a nonionic surfactant. Surfactants (also known as surface-active agents or emulsifiers) are essential to stabilize lipid nanoparticles dispersions and prevent particle agglomeration. Surfactants are amphipathic molecules that possess a hydrophilic moiety (polar) and a lipophilic moiety (non-polar), which together form the typical head and the tail of surfactants. At low concentrations, surfactants adsorb onto the surface of a system or interface. They reduce the surface or interfacial free energy and consequently reduce the surface or interfacial tension between the two phases [153]. The relative and effective proportions of these two moieties are reflected in Hydrophilic-Lipophilic Balance, or HLB value. The HLB scale, defined by Griffin, ranges in value from 1 to 20. Relative hydrophilicity increases with increasing HLB value. A value of 1 represents a strongly hydrophobic surfactant. Surfactants with HLB values in the range of 8–18 are suitable for the preparation of lipid nanoparticles through oil-in-water dispersion medium [154]. The surfactants used include ionic, nonionic and amphoteric surfactants. Ionic surfactants are traditionally thought to infer electrostatic stability, whilst nonionic surfactants are traditionally thought to infer steric repulsion stability [153]. Nonionic surfactants are preferred for oral and parenteral preparations over those with ionic properties because of their lower toxicity and irritancy. In general, the toxicity of surfactants decreases in the order cationic > anionic > nonionic > amphoteric [154]. 28 2.1.2.1. Hexadecane Hexadecane 99% was purchased from Sigma-Aldrich, and it was used as hydrophobic agent for osmotic stabilization of the lipid nanoparticles. Hexadecane, also called cetane, is a straight chain, saturated alkane hydrocarbon with molecular formula C16H34 and molecular weight 226.4 g/mol. Its melting point is 18ºC, thus is a liquid at room temperature with a density of 0.773 g/ml. Its research applications include the preparation of emulsions in water [157]. Its structure is shown in Figure 9. Figure 9- Structural formula of hexadecane 2.1.3. Bioactive Compounds 2.1.3.1. Rifampicin Rifampicin is a semisynthetic antibiotic derivative of rifamycin B and it was purchased from Sigma-Aldrich. Rifampicin is an orange-brown to red-brown crystalline powder very slightly soluble in water at neutral pH (2.5 mg/ml, 25ºC) [158]. It is soluble in dimethylsulfoxide (~100mg/mL), dimethylformamide, methanol (16 mg/ml, 25ºC), chloroform (349 mg/ml, 25°C), ethyl acetate (108 mg/ml, 25°C), and acetone (14 mg/ml, 25°C) [158]. Its molecular weight is 822.95 g/mol and its chemical formula is C43H58N4O12 [158]. Its structure is shown in Figure 10. Figure 10- Structural formula of rifampicin 29 2.1.3.2. Pyrazinamide Pyrazinamide, the pyrazine analogue of nicotinamide, is a white, crystalline powder, stable at room temperature and it was purchased from Sigma-Aldrich. Pyrazinamide is also known as pyrazinecarboxamide and its molecular weight is 123.11 g/mol [159]. Its chemical formula is C5H5N3O and its structural formula is shown in Figure 11 [159]. It is soluble in water (15 mg/mL) and chloroform, slightly soluble in ethanol (95%) and very slightly soluble in polar organic solvents [159]. Pyrazinamide is used to form polymeric copper complexes, create pyrazine carboxamide scaffolds useful as a component of mycobacteria identification kits. Figure 11 - Structural formula of pyrazinamide 2.1.3.3. β-carotene For this work, β-carotene was also selected as model lipophile. β-carotene delivery is of interest in the food industry, the nutraceutical industry, the skin care industry, and marketers of antioxidant medications. β-carotene possesses a molecular weight of 536.87 g/mol and a molecular formula of C40H56. It is a strongly-coloured red-orange pigment which belongs to a group of plant compounds called carotenoids. Carotenoids consist of an isoprene polymer backbone. β-carotene is composed of two retinyl groups, and is broken down in the mucosa of the small intestine to retinal, which is a form of vitamin A. β-carotene can be stored in the liver and converted in two molecules of vitamin A, being a provitamin. β-carotene is insoluble in water, making it an excellent model small molecule lipophile drug. Figure 12 shows its molecular structure. Figure 12 – Structural formula of β-carotene 30 2.1.4. Water The water used in all experiments was purified water by reverse osmosis and was obtained from a Milli-Q Plus, Millipore system. The water had an electrical conductivity of 0.51.5 μS/cm. 2.2. Methods 2.2.1. Preparation of Lipid Nanoparticles Lipid nanoparticles were prepared by ultrasonication and simple magnetic stirring methods. Both aqueous and lipid phases are separately prepared before mixing. The aqueous phase is composed of water (≥ 80.0% w/w), non-ionic surfactant (≤8.0% w/w) and hexadecane (≤3.5% w/w). Six formulations of empty SLNs was prepared ranging lipid content: SLN_1 represents the formulation with the lowest lipid phase content and SLN_6 represents the formulation with the highest lipid phase content. For NLC formulations was used a combination of natural oil with the MCFA. The content of bioactive compounds incorporated in lipid nanoparticles are shown at Table 3. Table 3 - Composition % (w/w) of bioactive compounds loaded to lipid nanoparticles. Formulation β RIF PYZ 2RIF_SLN - 0.06 - 2RIF_NLC - 0.06 - 4RIF_SLN - 0.11 - 4RIF_NLC - 0.11 - 7RIF_SLN - 0.20 - 7RIF_NLC - 0.20 - 4PYZ_SLN - - 0.11 4PYZ_NLC - - 0.11 β10-SLN 0.3 - - β10-NLC 0.3 - - In detail, the lipids are melted in a warm water bath above their melting points. Different temperatures conditions up than 50ºC were tested. When the MCFA or the MCFA/natural oil combination was fully melt, a hot aqueous solution with nonionic surfactant and hexadecane 31 preheated at the same conditions was added. A hot coarse o/w was obtained under magnetic stirring at 750 rpm for 1min and was subjected to sonication using a probe (MS72) sonicator (Bandelin, Germany) during 5 minutes. Hot o/w emulsion was allowed to cool to room temperature under magnetic stirring at 750 rpm to obtain the lipid nanoparticles. After 2h, the samples of each formulation were stored both in a refrigerator at 4ºC and at room temperature. For active agents-loaded lipid nanoparticles, the active agent was added to the lipid phase, 15 minutes earlier the addition of aqueous solution. Figure 13 shows the procedures. Figure 13 – Lipid nanoparticles production. Left: Lipid phase and aqueous phase in the warm water bath under same conditions (temperature and agitation). Top Right: Lipid phase (on the right) and aqueous phase (on the left) after weight. Middle Right: Addition of hot aqueous phase in melt lipid phase. Bottom Right: Sonication of hot pre-emulsion. 32 2.2.1.1. Lyophilisation of Lipid Nanoparticles Lyophilisation (or freeze-drying) was the final step of the production process. This process promotes water removal from a frozen sample, using sublimation and desorption under vacuum. Many pharmaceutical products, mainly heat sensitive compounds, are dried using this technique, since it improves the long-term physic-chemical stability and prevents degradation reactions such as hydrolysis. Lyophilisation is also used with nanoparticles to prevent particle aggregation. The aqueous samples of lipid nanoparticles were fast frozen under −80 °C in a deep freeze for 5h in ultra-low refrigerator. Then, the samples were moved to the freeze-drier (Christ Alpha 1-2 LD) during 48h in order to get powder of lipid nanoparticles. 2.2.2. Characterization of Lipid Nanoparticles 2.2.2.1. Particle Size In this work, a Zetasizer Nano ZS, Malvern Instruments (Malvern, UK) was used to measure the size of lipid nanoparticles produced. Particle size measurements were made in disposable cells at 25°C, by non-invasive back scatter, with dynamic light scattering detected at an angle of 173°. One ml of samples were used and 1:10 dilutions were performed (900μL ultrapure Milli-Q water + 100μL sample), to avoid multiple scattering phenomenon. The effective hydrodynamic diameter was calculated from the diffusion coefficient by the Stokes-Einstein equation (Eq.1) using the method of cumulants as an alternative to distribution analysis. In summary, the cumulants analysis gives a good description of the size that is comparable with other methods of analysis for spherical, reasonably narrow monomodal samples, with PDI value below 0.1. For samples with a slightly increased width, the Z-average size and PDI will give values that can be used for comparative purposes. All measurements were performed in triplicates with 20 runs by measurement. 2.2.2.2. Zeta Potential Zeta potential measurements were also performed using a Malvern Zetasizer Nano ZS, Malvern Instruments (Malvern, UK) at 25ºC. The samples were diluted 1:10 in ultrapure Milli-Q water and placed at a folded capillary cell (DTS1060) where an alternating voltage of ±150mV was applied. The measured electrophoretic mobilities were converted into zeta potential values using the Smoluchowski approximation. All measurements are performed in triplicate with 20 runs per measurement. 33 2.2.2.3. Particle Morphology Morphological observations of the SLNs and NLCs were performed using a transmission electron microscope (TEM). In this work, 20 µL of samples of with lipid nanoparticles previous analysed by DLS was deposited over a carbon covered TEM copper grid and then dried at room temperature. TEM images were collected using a microscope H-8100 Hitachi, operated with 200 kV of acceleration voltage incorporated with a CCD MegaView II bottom-mounted camera. 2.2.2.4. Encapsulation Efficiency An indirect method was used to determine the encapsulation efficiency of lipid nanoparticles using Eq.3 as explained in chapter 1. For all three active compounds loaded within lipid nanoparticles the same following process was used. After production, lipid nanoparticles were diluted and washed in milli-Q water. The dispersion was vortexed and then centrifuged at 12000 rpm for 30 min at 4ºC using centrifuge (Centrifuge 5810 R, Eppendorf). The supernatant was collected and the pellet was resuspended in fresh milli-Q water. This procedure was repeated three times. After washes, the amount of active agents in the supernatant was determined from its absorption using a double beam UV– VIS spectrophotometer (Model U-2000, Hitachi) and 1cm quartz cells. The amount of drug dissolved in supernatant can be quantified by spectrophotometer, since absorbance of a solution at a specific wavelength is proportional to the solute's concentration, as is expressed by Beer-Lambert's law: A= εlC Eq.4 where ε is the molar absorptivity (a constant that indicates how well the solute absorbs light of a particular wavelength, in units of M-1cm-1), l is the solution’s length the beam has to cross (1.0 cm for the quartz cell), and C is the solute’s concentration (mol/L). In this work, the measures of absorbance were done at 333 nm for rifampicin and at 548 nm for β-carotene. Pyrazinamide was analysed based on the formation of a coloured complex between the drug and sodium nitroprusside at alkaline pH with a mixing ratios of 4:1, which could be measured at 495nm (Figure 14). The solution of sodium nitroprusside at alkaline pH was prepared mixing a solution of 2% sodium nitroprusside in milli-Q water with a 2N sodium hydroxide solution in a ratio of 1:1 [160]. 34 Figure 14 – Pyrazinamide standard solutions used in constructing of the calibration curve, after reacting with alkaline sodium nitroprusside. Pyrazinamide concentration increases from left to right. In order to calculate the percentage of active compound incorporated is required to do a calibration curve by preparing several samples with well-known concentrations and measuring theirs corresponding absorbance at a particular wavelength. The resulted equation is then used to determine the mass concentration of the sample, through measuring the absorbance at same wavelength. Calibration curves of rifampicin, pyrazinamide and β-carotene were obtained by measuring solutions with known concentrations of the active compounds dissolved in milli-Q water. Samples were estimated in triplicates. Resulting calibration curves and respective equations are represented in Figure 15-17. Figure 15 - Rifampicin calibration curve 35 Figure 16 - β-carotene calibration curve Figure 17 - Pyrazinamide calibration curve 36 3. Results and Discussion 3.1. Study of Fabrication Parameters 3.1.1. Empty SLN The first objective of this work was to study SLNs at room temperature and their production conditions by miniemulsion using ultrasonication, based on a MCFA as solid lipid. The greatest advantage of this procedure is the easily production of SLNs without using organic solvents. In this work, a nonionic surfactant was employed, providing a steric hindrance, avoiding emulsion droplets from coming close to each other and thus preventing flocculation and coalescence. In this process, the MCFA is heated above its melting point. Aqueous solution of surfactant is mixed with lipid melt at the same temperature. Shear force from sonication will facilitate the formation of miniemulsion. The droplet size is governed by energy dissipation, temperature, surfactant and lipid concentration and other factors. The hot emulsion is then cooled down to form solid particles. Since the purpose of SLNs produced are for oral administration, the particles must be an average size less than 400 nm to easily cross intestinal cells [59]. Furthermore, the fabrication of particles comprises 100-200 nm it would be better, since it was reported particles smaller than 200 nm usually remain invisible to the reticulo-endothelial system (RES) and keep on circulation system over a prolonged period of time [35]. For this purpose, it was tested the influence of different parameters, such as applied lipid amount, temperature and sonication step on mean particle size expressed as Z-average mean size (Z-ave), polydispersity index (PDI) and zeta potential (ZP) (Table 4, Figure 18 and Figure 19) . Mean particle size of all formulations ranged from 69 ± 5 nm to 601 ± 100 nm and PDI from 0.87 ± 0.09 to 0.11 ± 0.02 (Table 4 and Figure 18). Since all the formulations shows a PDI higher than 0.1, this indicates that none are monodisperse. Finally, the ZP range from -35.8 ± 0.8 mV to -22.7 ± 0.7 mV (Table 4 and Figure 19), indicating good physical stability since particles aggregation was not likely to occur due to the electrostatic repulsion between the particles. For long-term stability a balance between electrostatic repulsion and steric effect of surfactant must be obtained. . 37 Table 4 - Physicochemical characteristics of empty SLN at different conditions (mean ± SD, n = 6). Non-sonicated T (ºC) a b c d Sonicated PdI ZP (mV) SLN_1 Z-Ave (d.nm) 601 ± 100 PdI ZP (mV) -29.6 ± 0.6 Z-Ave (d.nm) 146 ± 2 0.70 ± 0.08 0.18 ± 0.05 -25.8 ± 0.7 SLN_2 250 ± 6 0.56 ± 0.03 -27.5 ± 0.2 130 ± 9 0.18 ± 0.02 -27.8 ± 1.5 SLN_3 224 ± 28 0.46 ± 0.11 -28.8 ± 0.1 105 ± 1 0.18 ± 0.02 -27.8 ± 0.8 SLN_4 284 ± 19 0.87 ± 0.09 -30.4 ± 0.8 115 ± 1 0.18 ± 0.04 -29.4 ± 0.7 SLN_5 249 ± 16 0.70 ± 0.07 -29.3 ± 0.3 121 ± 3 0.15 ± 0.02 -28.6 ± 0.6 SLN_6 326 ± 6 0.85 ± 0.09 -32.4 ± 0.3 121 ± 1 0.18 ± 0.05 -28.1 ± 1.2 SLN_1 299 ± 8 0.55 ± 0.06 -31.0 ± 0.4 139 ± 2 0.17 ± 0.03 -27.2 ± 2.0 SLN_2 262 ± 13 0.44 ± 0.07 -29.9 ± 0.5 134 ± 23 0.18 ± 0.02 -25.1 ± 1.0 SLN_3 231 ± 25 0.54 ± 0.16 -28.3 ± 0.6 121 ± 25 0.20 ± 0.04 -30.3 ± 0.7 SLN_4 255 ± 8 0.47 ± 0.09 -30.0 ± 0.8 122 ± 10 0.17 ± 0.04 -28.6 ± 0.6 SLN_5 411 ± 22 0.51 ± 0.15 -26.5 ± 0.2 122 ± 11 0.19 ± 0.02 -27.2 ± 0.9 SLN_6 330 ± 55 0.45 ± 0.35 -35.8 ± 0.8 109 ± 19 0.18 ± 0.04 -31.7 ± 0.9 SLN_1 515 ± 140 0.52 ± 0.13 -30.6 ± 0.6 141 ± 1 0.14± 0.03 -28.4 ± 0.8 SLN_2 155 ± 4 0.26 ± 0.01 -28.3 ± 0.7 126 ± 7 0.15± 0.02 -26.8 ± 0.9 Formulation SLN_3 140 ± 1 0.20 ± 0.01 -29.3 ± 0.6 97 ± 30 0.23 ± 0.09 -28.9 ± 1.0 SLN_4 196 ± 2 0.30 ± 0.03 -31.5 ± 0.2 119 ± 1 0.13 ± 0.02 -29.2 ± 0.6 SLN_5 149 ± 1 0.29 ± 0.02 -29.9 ± 0.5 120 ± 1 0.17 ± 0.05 -28.5 ± 3.9 SLN_6 179 ± 1 0.25 ± 0.01 -25.5 ± 0.7 122 ± 2 0.18 ± 0.01 -28.2 ± 0.7 SLN_1 333 ± 50 0.41 ± 0.03 -30.0 ± 0.5 139 ± 1 0.11 ± 0.02 -26.6 ± 0.6 SLN_2 93 ± 7 0.30 ± 0.08 -29.1 ± 0.6 87 ± 12 0.21 ± 0.02 -22.7 ± 0.7 SLN_3 144 ± 44 0.87 ± 0.05 -12.3 ± 0.6 105 ± 1 0.23 ± 0.03 -28.4 ± 0.6 SLN_4 90 ± 3 0.46 ± 0.03 -29.3 ± 0.3 69 ± 5 0.19 ± 0.03 -29.4 ± 0.7 SLN_5 99 ± 2 0.49 ± 0.05 -34.7± 1.5 97 ± 30 0.23 ± 0.09 -28.9 ± 1.0 SLN_6 121 ± 2 0.40 ± 0.02 -30.6 ± 0.3 126 ± 7 0.19 ± 0.03 -26.0 ± 0.2 38 Figure 18- Z-ave and PDI of empty SLNs with different content of MCFA (SLN_1 represents the lowest lipid phase content and SLN_6 represents the highest lipid phase content), ranging from a lowest temperature (a) to highest temperature (d). 39 Figure 19 - Zeta Potential of empty SLNs with different content of MCFA (SLN_1 represents the lowest lipid phase content and SLN_6 represents the highest lipid phase content), ranging from a lowest temperature (a) to highest temperature (d). 40 3.1.1.1. Influence of Temperature The effect of temperature on pre-emulsion was investigated. Four different temperatures up than 50ºC were applied. The main influence of temperature was observed on non-sonicated samples. Table 4 shows a notorious size reduction of non-sonicated particles with the increase of temperature, while PDI and ZP values remain with non-significant variation. These results are expected since in general, the increase of temperature usually decreases the viscosity of the lipid and aqueous phases and hence the efficiency of the agitation and mixture is increased. In other words, increasing the fusion temperature of the lipids results in smaller droplet/particle sizes [113]. 3.1.1.2. Influence of Lipid Content The influence of MCFA content on SLNs properties were also investigated (Table 4 and Figure 18-19). From analysis of non-sonicated samples in a, c and d of Figure 18, it seems evident that particle size of SLNs prepared with the lowest concentration of MCFA are larger when compared with other SLNs. In addition, PDI of these samples are also high. However, ZP appears not be affected as well as all features considered of lipid particles obtained by sonication. As PDI of non-sonicated SLNs produced with lowest lipid content are also very high, the Ostwald ripening effects could be an explanation for their larger sizes. Ostwald ripening is a thermodynamically driven process, in which smaller particles dissolve and redeposit onto the surface of larger particles. This process occurs because smaller particles have larger surface area and higher surface energy and hence higher Gibbs free energy than the larger particles. All systems tend to attain lowest Gibbs free energy. In other words, larger particles are more energetically stable and thus, preferred over smaller particles. Ostwald ripening can be reduced by minimizing PDI in the particle size but it cannot be prevented. In conclusion, although in general low lipid content decreases the aggregation phenomenon, it may also increase the Ostwald ripening effects and causes the migration of surfactant molecules from the particle interface to aqueous medium decreasing the electrostatic repulsive force and leading particle aggregation. 41 3.1.1.3. Influence of Sonication The influence of sonication on SLNs features was represented in Table 4 and Figures 18-19. Figure 20 - Macroscopic aspect of empty SLNs: (left) - non-sonicated sample; (right) – sonicated sample Comparing the macroscopic aspect between non-sonicated and sonicated SLNs suspension (Figure 20), it is possible to see that non-sonicated SLNs dispersion has a milky aspect, while sonicated SLNs dispersion are more clear and bluish. From literature, usually colloidal dispersions with droplet sizes between 20–100 nm are transparent or bluish, while colloidal dispersions with larger droplets sizes up to 500 nm has a milky aspect [107]. Thus, through macroscopic analysis, it can be expected a decreased on mean SLNs particle size of sonicated samples. The particle size reduction is confirmed by DLS results. At Figure 18, it is clear the effect of sonication in both Z-ave and PDI of MCFA SLNs. All sonicated SLNs show a mean Z-ave lower than 150 nm. This is due to shear stress, which is resultant from the application of ultrasound energy that promotes a shear on MCFA molecules, impairing aggregation, thus, avoiding the formation of larger particles. Despite of this significant effect on particle size, ultrasonication does not appear to play an essential role in physical stability of SLNs, since ZP is very similar between non-sonicated and sonicated SLNs, although there is an insignificant decrease in absolute ZP value (Figure 19). In conclusion, the application of ultrasound energy seems to decrease the mean SLNs particle size and also homogenize the SLNs in suspension, since PDI values decrease when sonication is applied. 42 3.1.1.4. Lyophilisation Figure 21 – Powder resulting of SLN_1b_II lyophilisation Lyophilisation was applied to some SLNs samples in order to remove the excess water and also to enhance stability of powder SLNs, since Ostwald ripening can be avoided and particles aggregation decreased. The obtained powder of SLN_1b_II is shown at Figure 21. Table 5 presents the Z-ave, PDI and ZP of the SLN_1b_II before and after lyophilisation. Z-ave of lyophilised SLNs is slightly larger when compared to liquid dispersion, increasing from 153 ± 31 nm to 191 ± 4 nm. However, PDI of lyophilised SLNs increased from 0.13 ± 0.04 to 0.49 ± 0.10, resulting on a sample far polydisperse. Examining particle size distribution of SLN_1b_II before and after lyophilisation (Figure 22), it is possible to realize the appearance of a second particles population larger than 1 µm in reconstituted lyophilised sample. This larger population could be result of aggregation phenomenon, indicating physical instability of SLNs and explaining the increase of PDI. However, contrary as expected, absolute ZP value increased from 30 ± 2 mV to 35 ± 2 mV, meaning an increase on particles stability. An explanation for these results could be an insufficient stirring force/time applied on resuspension of lyophilised SLNs in milli-Q water. If the attractive forces holding the aggregate together are very large compared with the typical forces used in stirring, mixing or ultrasonic probes, the system is regarded as permanently aggregated. Thus, the larger population of SLNs on lyophilised SLN_1b_II could be a result of flocculation. To explore this hypothesis, it could be done a comparative study between different forces used to resuspension of lyophilised SLNs. In addition, it could be investigated the combination of a cryoprotector previously to lyophilisation since it was found that cryoprotective agents preserve the physicochemical properties of SLNs and decrease SLNs aggregation. Typical cryoprotectors are sorbitol, mannose, trehalose, glucose, and polyvinylpyrrolidone. The best results occurred when cryoprotector is in concentrations of 10-15% [162]. 43 Table 5 – Physicochemical characteristics of the SLN_1b_II. Values are mean ± SD, n = 3 Formulation Z-Average (nm) PDI ZP (mV) SLN_1b_II 153 ± 32 0.13 ± 0.04 -30 ± 2 Lyophilised SLN_1b_II 191 ± 4 0.49 ± 0.10 -35 ± 2 Figure 22 – Particle Size Distribution by Intensity of SLN_1b_II before and after lyophilisation. 3.1.1.5. Long-Term Stability In this work, SLNs formulations were stored at room temperature (25ºC) and in the refrigerator (4ºC), during seven months. The long-term stability of both formulations was assessed by means of particle size (Z-ave and PDI) and ZP measurements. The stability was also monitored by macroscopic observations. The samples stored at room temperature remained with a milky-like appearance, although some of samples show particle sedimentation. However, during storage for more than one week some unpredictable gelation occurred in SLNs containing samples stored at 4ºC. Gels are more structured than liquids. SLNs are able to organize themselves in superstructures. The change in morphology of lipid nanoparticles from spheres to platelets is responsible for the gelation of SLN dispersions [163]. Depending on the composition, especially of the emulsifier(s) and the amount of lipid matrix, a gelation of the normally liquid dispersions can be observed on storage [163]. Table 6 shows the obtained Z-ave, PDI and ZP results for SLN_4b_I, an example of a macroscopic milky-like colloidal SLNs dispersion stored at room temperature. From results analysis, it can notice a decrease in the absolute ZP value from 39.1 ± 0.9 mV to 28.3 ± 0.6 mV, indicating changes in the particles structure. This can be confirmed by the DLS measurements where particle size growth was observed (Table 6 and Figure 23). The mean particle size 44 remained lower than 400 nm, although there was an increasing on the size from 154 ± 25 nm to 223 ± 54 nm. Since PDI and particle size remain relatively low, homogeneously sized SLNs displayed a satisfactory long-term stability. Table 6 - Physicochemical characteristics of the SLN_4b_I. Values are mean ± SD, n = 3. Formulation SLN_4b_I SLN_4b_I after 7months Z-Average (nm) PDI ZP (mV) 154 ± 25 0.07 ± 0.03 -39.1 ± 0.9 223 ± 54 0.05 ± 0.02 -28.3 ± 0.6 Figure 23 - Particle Size Distribution by Intensity of SLN_4b_I after one day and 7 months after production. The shape and surface morphology of empty SLNs after seven months were also analysed by TEM and the resulting images are represented in Figure 24. In the TEM study, the size of the SLNs was found to be in agreement with the DLS. All the particles were found to be smoothly spherical or oval in shape with a well-defined periphery. No obvious aggregation of the SLNs was observed. It should be noted, however, that the particles align in three dimensions, whereas the micrographs only allow particle presentation in two dimensions so that the given particle dimensions are only estimates due to the fact that a particle observation exactly top-on and edge-on, respectively, is rare and cannot be discriminated from slight particle twists, in addition. 45 Figure 24 – TEM image of SLN_4b_I after 7 months. 3.1.2. Empty NLC For NLC production, the fabrication procedure was similar to the SLNs fabrication and was described in chapter 2. Table 7 presents the Z-ave, PDI and ZP of the NLC. The Z-ave of NLC is larger when compared to SLN_4b. Since both lipid nanoparticles have an equal percentage of lipid content and were produced at same conditions, the increase of the size from 119 ± 0 nm to 282 ± 56 nm are related with the use of natural oil. The combination of different chain fatty acid triglycerides promotes changes in structure of lipid nanoparticles. These changes on particles surface are also supported by the slightly reduction of absolute ZP value from -29.2 ± 0.6 mV to -26.4 ± 0.7 mV. In conclusion, despite the growth of particle size, NLC size remains lower than 400 nm, as required to easily cross intestinal cells. Table 7 – Physicochemical characteristics of the empty NLC. Values are mean ± SD, n = 3. Formulation NLC Z-Average (nm) 282 ± 56 PDI 0.43 ± 0.09 ZP (mV) -26.4 ± 0.7 46 3.2. Bioactive Compounds Loaded SLNs and NLCs The second main goal of this project was to investigate the entrapment efficiency of some bioactive compounds in MCFA based lipid nanoparticles. The anti-TB drugs rifampicin and pyrazinamide were chosen to encapsulate within lipid nanoparticles and β-carotene was also used as lipophilic compound model. From the first part of this study, it had to be considered a compromise between the adequate size, a low PDI and also a good stability of the samples for the final choice of the parameters to be used in loaded lipid nanoparticles production. As mentioned above, the desired formulations have to be lower than 400 nm. Furthermore, the fabrication of particles comprises 100-200 nm it would be better, since it was reported particles smaller than 200 nm usually remain invisible to the reticulo-endothelial system (RES) and keep on circulation system over a prolonged period of time [35]. Taking all this considerations into account, the final parameters chosen to lipid nanoparticles production were those that correspond to the SLN_4 formulation and the temperature of lipid melting must be up 60ºC, followed by 5 minutes of sonication. Optimal temperature also depends of drug characteristics. Figure 25 shows the difference in macroscopic appearance of empty and loaded NLCs before and after sonication. After sonication, all the dispersions are significantly more transparent. Figure 25 – Differences in macroscopic appearance of empty and loaded NLCs immediately before and after sonication. 47 3.2.1. β-carotene β-carotene was incorporated in SLNs and NLCs. The Z-ave, PDI and ZP of loaded particles were measured one day after production and the results are represented in Table 8. The %EE of the prepared lipid nanoparticles were determined indirectly, by calculating the amount of free β-carotene (non-encapsulated) present in the aqueous phase of dispersions, as explained in chapter 2 and was also represented in Table 8. As expected, SLNs and NLCs loaded with β-carotene are larger when compared with empty SLNs (Table 4) and empty NLCs (Table 7). Particle sizes remain lower than 400 nm, as required. No important changes seem to occur on stability of both loaded SLNs and NLCs, since ZP values remain similar from empty SLNs and NLCs. In addition, comparing with rifampicin (Table 9) and pyrazinamide (Table 10) loaded-particles, β-carotene shows the highest percentage of encapsulation efficiency. This result was predictable, since β-carotene is the most lipophilic of these three active compounds. Also, since β-carotene is less soluble in oil then in fat, the %EE of β10-SLN is higher than β10NLC. Table 8 - Physicochemical characteristics of the β-carotene lipid nanoparticles. Values are mean ± SD, n = 3. Formulation Z-Average (nm) PDI ZP EE (mV) (%) β10-SLN 281 ± 19 0.34 ± 0.03 -29 ± 5 95 ± 5 β10-NLC 328 ± 65 0.28 ± 0.06 -25 ± 3 83 ± 6 Morphological analysis of β-NLC was performed by TEM and the resulting images are represented in Figure 26. The size of the particles was in agreement with DLS results. TEM revealed nanoparticles with almost spherical shapes and a roughness surface. However, it was also reported the presence of some aggregates and some particles with an irregular shape. Figure 26- TEM image of β-carotene loaded NLC. 48 3.2.2. Rifampicin The Z-ave, PDI, ZP and %EE of RIF loaded lipid nanoparticles are presented in Table 9. As expected RIF-NLCs samples are larger than RIF-SLNs. In addition, RIF loaded particles show a reduction in the electrical charge at the surface to mean values below -20 mV. These results could be taken as an indication that RIF is entrapped in the lipid matrix of lipid nanoparticles. Due to the high solubility of RIF in the lipid core, the encapsulation efficiency was good enough for all the formulations, ranging between 67 ± 7% and 85 ± 5%. In literature, it was found that NLCs can encapsulate slightly more amount of drug than SLNs. However, none significant differences in %EE it seems to exist between RIF-SLNs and RIF-NLCs. Muller et al. also reported that the presence of liquid lipid in the solid matrix avoids the drug expulsion during storage that can occur when the lipid matrix undergoes polymorphic transformations from unstable to more stable configurations [28]. In this work, no long-term stability study was done to verify this event. And, since, it is obvious that polymorphic crystal transformation during storage can affect the encapsulation efficiency and the drug release profile of lipid nanoparticles, additional thermal and crystallization studies must be done in order to a better control of encapsulation efficiency and the drug release. Table 9 - Physicochemical characteristics of the rifampicin lipid nanoparticles. Values are mean ± SD, n = 3. Formulation Z-Average (nm) PDI ZP EE (mV) (%) 2RIF_SLN 341 ± 78 0.45 ± 0.12 -20.3 ± 2.2 74 ± 10 2RIF_NLC 391 ± 210 0.62 ± 0.10 -18.5 ± 1.3 67 ± 7 4RIF_SLN 84 ± 1 0.23 ± 0.01 -17.8 ± 0.7 69 ± 1 4RIF_NLC 180 ± 90 0.36 ± 0.08 -19.5 ± 0.9 69 ± 1 7RIF_SLN 181 ± 31 0.34 ± 0.03 -19.2 ± 1.3 79 ± 7 7RIF_NLC 215 ± 5 0.28 ± 0.03 -18.8 ± 0.6 85 ± 5 From TEM analysis represented at Figure 27, it can be observed that RIF-NLCs also exhibit spherical shapes, which indicates drug loading is not lead to morphological changes. Again, the particle size was in concordance with particle size analysis data obtained from dynamic light scattering 49 Figure 27 - TEM image of rifampicin loaded NLC. In literature, it was found two studies involving the encapsulation of RIF in lipid nanoparticles. In first one, RIF was loaded into Compritol ATO 888 SLNs fabricated by a modified microemulsion technique. The particles sizes obtained were 141 ± 13nm, ZP value was −3.5 ± 0.8mV and %EE was 65 ± 3% [164]. In second one, RIF was encapsulated in group with pyrazinamide and isoniazid in a stearic acid SLNs produced by emulsion solvent diffusion with a %EE of 51 ± 5% [89]. Perhaps due to RIF was been encapsulated combined with other anti-TB drugs the %EE was significant lower than results described in this work. In general, results obtained in this project seem to be very good, by comparison with reported results by literature. For one hand, it was obtained higher encapsulation efficiency and for another hand, fabrication method applied is free-use of organic solvent, meaning less toxicity of the obtained particles. 3.2.3. Pyrazinamide The results of Z-ave, PDI, ZP and %EE of pyrazinamide loaded lipid nanoparticles are represented in Table 10. The %EE value obtained range from 14 ± 8% for PYZ-SLN to 29±15% for PYZ-NLC. These low results are expected since pyrazinamide is the most hydrophilic compound in that study, being soluble in water (15 mg/ml). Thus, pyrazinamide partitioned between the melted lipid and aqueous phase. However, in literature pyrazinamide was encapsulated in a stearic acid SLNs combined with isoniazid and rifampicin and %EE reported was 41 ± 7% [89]. Since pyrazinamide is one of 50 anti-TB drugs administered in current chemotherapy, in further studies could be investigated the combine incorporation of pyrazinamide with other anti-TB drugs such as rifampicin, isoniazid or ethambutol. Furthermore, the process of cooling from high to room temperature could be optimized in order to control crystallization processes and consequently the encapsulation efficiency. Table 10 - Physicochemical characteristics of the pyrazinamide lipid nanoparticles. Values are mean ± SD, n = 3. Formulation Z-Average (nm) PDI ZP EE (mV) (%) 4PYZ-SLN 217 ± 87 0.31 ± 0.08 -21.0 ± 0.7 14 ± 8 4PYZ-NLC 313 ± 214 0.34 ± 0.17 -22.7 ± 2.4 29 ± 15 The resulting images of TEM analysis of PYZ-NLC are represented in Figure 28. The size of the particles was in agreement with DLS results. However, TEM revealed nanoparticles with poorly spherical and irregular shapes. It was also reported the presence of a lot of aggregates, indicating important alterations on PYZ loaded particles structure. Figure 28 - TEM image of pyrazinamide loaded NLC 51 4. Conclusions and Further Works In the present work MCFA SLNs and NCLs with a combination of MCFA and natural oil as lipid phase were successfully prepared by sonication technique. This technique was simple, reproducible, prepared nanoparticles without the need of organic solvents or any sophisticated instruments and has the potential to easily scale up for large scale production. Furthermore, the lipid nanoparticles obtained in the present work are suitable carrier systems for the incorporation of different bioactive compounds intended for oral administration, since mean particle size of all formulations ranged from 69 ± 5 nm to 601 ± 100 nm and the great part of the formulations revealed a particle size less than 400 nm which easily cross intestinal cells. The presence of a small quantity of bigger particles was not seen as critical, because the preparations were intended for oral use and not for intravenous administration. Also, it was obtained a lot of formulations with a particle size less than 200 nm, which is an even better result since they will remain invisible to the reticulo-endothelial system (RES) and keep on circulation system over a prolonged period of time. The Zeta Potential obtained was negatively enough to ensure a good physical stability of the particles, indicating that the nonionic surfactant used is a good surfactant for lipid nanoparticles produced with this particular formulation. In general, the formulations show a PDI higher than 0.1, indicating that none are monodisperse. To obtain formulations with lower PDI values, such that they could be considered monodisperse, filtration could be done with porous of low diameters, such as 450 nm or even 200 nm. In industry, other methods to produce lipid nanoparticles could be used, and some of them, like high pressure homogenization, will naturally produce monodisperse populations of particles. Morphological studies using TEM images showed spherical to oval SLNs and RIF-NLCs with well-defined periphery. The particle size was in concordance with particle size analysis data obtained from dynamic light scattering. However, the β-NLCs and PYZ-NLCs show an irregular shape and some tendency to aggregate. Lyophilisation of lipid nanoparticles seems to be a good alternative to remove excess of water and to enhance stability of the particles during the storage. Also, the obtained powders can be used to produce classic solid dosage forms, can be redispersed in water or juice prior to administration, and can also be used for the filling of hard gelatine capsules. However, further studies must be done to prevent aggregation of lyophilised particles. It could be explored the use of an additional cryoprotector previously lyophilisation. Furthermore, other alternatives to lyophilisation could be investigated such spray drying. This method has been used scarcely for SLNs formulation, although it is cheaper than lyophilisation Rifampicin, Pyrazinamide and β-carotene were successful loaded at lipid nanoparticles. Rifampicin encapsulation efficiencies obtained in this work were higher than the values reported in literature for encapsulation of rifampicin in lipid nanoparticles. Pyrazinamide shows low encapsulation efficiencies due to its hydrophilicity leading to partition between lipid phase and 52 aqueous phase. However, pyrazinamide could be encapsulated combined with other anti-TB or could be done a stearic modification in order to improve its entrapment into solid matrix. In further studies could be explored the encapsulation of isoniazid and ethambutol in produced lipid nanoparticles. Nevertheless, further analysis such as DSC must be performed to characterize the state and the degree of crystallinity of lipid dispersions provide guidance to better control polymorphic transition of lipid nanoparticles, which have profound impact on increasing loading capacity, encapsulation efficiency, manipulating release pattern and even improving long term stability of lipid nanoparticles. 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