Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs

Mestrado em Engenharia Biomédica Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs Master Thesis developed in the course of Dissertation RICARDO LEANDRO DELINDRO RIBEIRO Supervisor PROFESSOR MARIA DE LA SALETTE REIS JULY 2013 Para a Sílvia. Para o Daniel. Acknowledgements For different reasons, this work would not have been possible without the support of a number of high quality individuals, surrounded by which I found myself lucky to be most of the time. I would like to start by thanking my thesis supervisor, Salette Reis, first of all for having accepted me in her group and having trusted me with this work, but also for all the scientific, material and moral support, and mainly for making sure that everyone in the group treated me well. It must have been a hard task, I am sure. Secondly, I would like to thank Marina Pinheiro, for all the guidance and patience in this work, (even through the rough times, the bad results, and the dangerously close deadlines), and for always being present when needed. This work would not be what it is without her. I am in debt to a few ones that helped me with some experimental techniques: Fernanda Andrade, for the help with the MTT assays; José das Neves, for the help with the HPLC measurements; and Ana Cardoso, for the help with the DSC experiment. I would also like to thank the ones who I shared the laboratory with, and who also shared with me their knowledge, experience and overall good mood. They told me not to write their names, but I'm not going to obey: to Catarina, Catarina, Catarina, Joana, Miriam and Nini, but also to dona Manuela, Patrícia, and Sofia. A special and warm thank you note goes out to Júlia, for all these years of healthy partnership, and for always laughing, laughing out loud. To all my colleges in the MEB programme, specially to Raquel Almeida and André Carvalho, for all those lunatic lunches and (i)rational conversations. A deep acknowledgement goes naturally to my parents, which are, and always will be, present in everything I do. Finally, I would like to thank those two individuals, homo sapiens of the highest quality, that complete my life, and to whom this work is dedicated. To Sílvia, for always being there, for always supporting my choices, for being patient with my doubts, and, above all, for not having run away. And to Daniel, for having born at exactly the right moment. i Abstract Tuberculosis (TB) is still an ongoing public health concern in African, Asian and South American countries, where it still has a strong prevalence, resulting in a heavy economic, social and human burden. In 2011, the World Health Organization (WHO) has reported an estimated 1.4 million deaths due to TB, a disease caused by the infection of Mycobacterium tuberculosis (MTb). There have been determined efforts to fight this disease, and the search for new antitubercular drugs plays a crucial role. In spite of these efforts, the most recent drug in the market dates back 50 years, and so new delivery strategies that improve the efficacy of existing treatments may become important in this fight. The goal of this work was to develop a nanocarrier system for the delivery of antitubercular drugs. The chosen nanocarriers were lipid nanoparticles, more specifically nanostructured lipid conjugates (NLCs). These particles were loaded with two anti-tubercular drugs: rifampicin (RIF) and rifabutin (RFB). Since the lung is the primary site of infection in TB, the proposed route of administration for this strategy is the pulmonary route. Once inhaled, the particles should be able to travel to the pulmonary alveoli and reach the alveolar macrophages (AMs). Produced particles thus must have an appropriate size, otherwise they will be trapped in the upper airways or leave the lung on exhaling. Also, it is known that AMs have specific receptors that bind to sugars. Surface modification by mannose coating was performed to take advantage of these receptors and improve cellular uptake by AMs. The developed particles were characterized in terms of size, zeta and morphology. Results showed particles with size and morphology suitable to reach the pulmonary alveoli, and loading efficiency for both drugs was above 80%. The success of mannose coating was confirmed by FTIR analysis. Cytotoxicity of the formulation was evaluated by MTT assay with three different cell lines. Although more studies are definitely needed, the results from the present work pose a strong argument for NLCs as a promising strategy for the pulmonary delivery of antitubercular drugs. iii Resumo A tuberculose (TB) apresenta-se ainda como um problema de saúde pública considerável em países Africanos, Asiáticos e Sul Americanos, onde ainda tem uma elevada prevalência, resultando num pesado fardo económico, social e humano. Em 2011, a Organização Mundial de Saúde estimou que a TB terá sido responsável, em todo o mundo, por 1.4 milhões de mortes. A TB é uma doença causada pela infeção por Mycobacterium tuberculosis. Inúmeros esforços têm sido concentrados no combate a esta doença, e a pesquisa por novos fármacos tem, aqui, um papel preponderante. No entanto, e apesar destes esforços, o mais recente fármaco para o combate à TB tem já 50 anos. Novas estratégias de transporte e libertação de fármacos, que melhorem a eficácia dos tratamentos já existentes, poderão tornar-se importantes nesta luta. O objectivo deste trabalho foi desenvolver um sistema de nanopartículas para o transporte e libertação de fármacos de combate à TB. As nanopartículas escolhidas foram nanopartículas lipídicas, mais especificamente partículas lipídicas nanoestruturadas (NLC). Nestas foram introduzidos dois fármacos: rifampicina (RIF) e rifabutina (RFB). Dado que os pulmões são o principal foco de infecção por TB, a via de administração proposta é a inalatória. Uma vez inaladas, as NLC deverão depositar-se nos alvéolos pulmonares, onde se encontram os macrófagos alveolares (AMs). As partículas produzidas deverão, portanto, ter um tamanho apropriado a este objectivo. É também conhecido que os AMs têm receptores de açúcares específicos. A superfície das partículas foi então modificada para expor moléculas de manose, com o ojbectivo de aumentar a As nanopartículas desenvolvidas foram caracterizadas em termos de tamanho, potencial zeta e morfologia. Os resultados revelaram partículas com tamanho e morfologia adequados para atingir os alvéolos pulmonares. A taxa de incorporação para ambos os fármacos foi acima de 80%. A modificação da superfície com manose foi confirmada por análise FTIR. A citotoxicidade foi avaliada por ensaios de MTT, com três linhas celulares. Mais estudos são definitivamente necessários, mas os resultados do presente trabalho apresentam um forte argumento a favor da utilização de NLC como uma promissora estratégia para a administração pulmonar de fármacos no combate à TB. v Abbreviations and symbols AM Alveolar macrophage CFC Chlorofluorocarbon CIP Ciprofloxacin DCP Dicetylphosphate DMEM Dulbecco`s modified eagle medium DPI Dry powder inhaler DPPC Dipalmitoylphosphatidylcholine DSC Differential scanning calorimetry EPC Egg phosphatidylcholine FTIR Fourier transform infra-red HIV Human immunodeficiency virus IC50 Half maximum inhibitory concentration LEV Levfloxacin LHLN 6-lauroxyhexyl lysinate MBSA Maleylated bovine derum albumine MDI Metered-dose inhaler MDR Multi drug resistant MTb Mycobacterium tuberculosis NLC Nanostructured lipid carrier NP Nanoparticle O-SAP O-steroyl amylopectin OFX Ofloxacin PBS Phosphate buffer saline PDI Polydispersity index PEG Polyethylene glycol PLGA Poly(lactide-co-glycolide) acid pMDI Pressurized metered-dose inhaler PC Phosphatidylcholine PS Pulmonary surfactant RFB Rifabutin RIF Rifampicin SEM Scanning electron microscopy SLN Solid lipid nanoparticle TB Tuberculosis WHO World Health Organization XDR Extremely drug resistant ζ Zeta xiii 1 Introduction 1.1 Motivation Tuberculosis (TB) is still far from being a health concern of the past. Although less frequent in European countries and North America, it has a strong prevalence in Africa, Asia and South America. In 2011, the World Health Organization (WHO) reported an estimated 8.7 million new cases and 1.4 million deaths from TB, thus making it the second leading cause of death by infectious diseases in the world. To address this heavy public health burden, in 2006 the WHO lauched the Stop TB Strategy. The goals of this strategy are, for 2015, to reduce prevalence of and deaths due to TB by 50% compared with a baseline of 1990, and for 2050, to eliminate TB as a public health problem [1]. The search for new anti-TB drugs is, of course, of key importance in this fight, but notwithstanding this search, 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 [2]. This constituted the major motivation factor behind this work. It's main goal was the development of a new delivery strategy for the treatment of TB, through the use of lipid nanoparticles as carriers for two rifamycins (i.e. rifampicin and rifabutin), commonly used as anti-TB drugs. The aim of this project was to produce a nanosystem for pulmonary administration, featuring both passive and active targeting strategies, in order to improve drug uptake by alveolar macrophages. 1.2 Characterization of TB TB is a disease caused by the infection of Mycobacterium tuberculosis (MTb). It can affect practically all organs of the human body, but the lung (pulmonary TB) is of particular high incidence. This is to be expected, since the infection starts with the inhalation of bacilli of MTb during breathing, leading the bacteria directly to the lung. Due to their size, the bacilli are able to reach the pulmonary alveoli, where they are phagocyted by the alveolar macrophages (AMs) [3] (Figure 1). 1 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs Inside the AMs, the bacilli reside in a membrane-bound vacuole, and for this reason some are able to avoid fusion with lysosomes and posterior digestion [4], ending up co-existing with the AMs [5]. 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 [6](Figure 2). Moreover, MTb 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. The estimates are that one third of the world's population is infected with the organism, although usually the infection is present in its dormant state [7]. Some symptoms may be associated with pulmonary TB and extra pulmonary TB, and they could be of help when diagnosing the disease. In pulmonary TB, symptoms include cough, production of sputum in later stages (due to inflammation and tissue necrosis), hemoptysis (only in rare cases), pleuritic pain, dyspnea (unusual, unless there is extensive disease), and may also cause severe respiratory failure. X-ray of the lung and examination of sputum is often used to confirm pulmonary TB. Extra pulmonary TB has a wider range of symptoms, depending on which organ is 2 Figure 1: Contagion and infection by MTb Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs affected, and in many cases infection produces systemic effects, rather than local ones. Moreover, these effects are many times associated with other ailments, such as human immunodeficiency virus (HIV) infection, diabetes mellitus, and neoplastic diseases, which considerably delays diagnosis and increases misdiagnoses, specially with patients co-infected with HIV [8]. 1.3 Traditional chemotherapy Treatment for TB almost always involves a cocktail of drugs administrated through long periods of time, which contributes to patient non-compliance, resulting in multi drug resistant (MDR), extremely drug resistant (XDR) [1], and even totally drug resistant strains of TB, which are considerably harder to treat [9] (Figure 3). Also, progress on new drug therapies has been developing slowly, and the most recent of anti-TB drugs currently in use dates back 50 years. Sarkar et al., in their review of the present TB chemotherapy available and of new and emerging drugs, stressed how essential further research in a new drug target is to fight MDR and XDR TB [2]. Currently available chemotherapy includes first-line drugs, such as isoniazid, pyrazinamide, rifampicin, and ethambutol, and second-line drugs, such as para-aminosalicylic acid, ciprofloxacin/ofloxacin, clofazimine, cycloserine, ethionamide, rifabutin, streptomycin, and thioacetazone [10]. These second-line drugs are only used when treatment with first-line drugs fails. They are less effective, more toxic, and unavailable in many countries due to high costs [11]. The two drugs used in the present work were rifampicin (RIF) and rifabutin (RFB). They belong to the family of rifamycin antibiotics, which are among the most potent anti-tuberculosis agents known. They possess a unique ansa structure consisting of an aromatic nucleus linked on both sides by an aliphatic bridge [12]. RIF is a red crystalline powder. It exhibits a half life between 2.3 and 5 hours on initiation of therapy, but this value decreases to between 2 and 3 hours after repeated treatment. Rifabutin (RFB) is a violet crystalline powder. It has a longest half life, between 32 and 67 hours, 3 Figure 2: Extrapulmonary TB. Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs but it also shows increased toxicity, and adverse effects include rash, gastrointestinal disturbance, neutropenia, and occasional uveitis [10]. There are several new drug candidates currently in research and in clinical trials, and several existing drugs are in a state of re-evaluation [9]. In December 2012, the FDA granted an accelerated approval of a new drug, bedaquiline, but only as part of a combination therapy to treat adults with MDR TB when other alternatives are not available [13]. However, bedaquiline has not yet gone through a phase III trial, and several accounts of heart failure have been reported, which may result in bedaquiline being removed from the market. This illustrates how important it is to find new strategies to fight this disease. 1.4 Outline of the dissertation The present dissertation is divided in five chapters. Chapter 1, the present chapter, constitutes a brief introduction to the theme and the main goals of the dissertation. Chapter 2 presents some scientific considerations of TB and it's current treatment options, and it ends with a state of the art regarding nanosystems as carriers for anti-TB drugs. This state of the art, with appropriate modifications, is intended to be submitted for publication as a review article. In Chapter 3, the materials and methods used in the present work are presented and explained. Results from the experimental work are shown and discussed in Chapter 4. Finally, Chapter 5 constitutes an overall reflection on the goals and achievements of this work. It also tries to outline possible paths for future work. 4 Figure 3: Problems associated with traditional TB chemotherapy. 2 Nanosystems for the pulmonary delivery of anti-tuberculosis drugs Since the lung is the most important point of access in the case of infection by MTb [14], [15], exploiting the inhalatory route for drug delivery becomes an exciting hypothesis to fight the disease [16]–[18]. Indeed, the lung is the ideal target site for anti-TB drug delivery, and could provide a delivery portal requiring smaller doses for efficacy, exhibiting reduced toxicity and fewer side effects [3]. Also, the respiratory system behaves as an “aerosol filter”, a property that can be exploited to target particles having specific attributes to the lung [15], and since the lung mucosa has a large surface from which drugs may be systemically absorbed into the bloodstream, escaping the first-pass metabolism [14], enhancing overall bioavailability. This makes pulmonary delivery of drugs an interesting approach for the treatment of pulmonary infections. Adding to this, as was described in section 1.2, pathogenic TB bacilli establish infection mainly in alveolar macrophages [16]. In this regard, it would be of interest not only to deliver the drugs to the lung, but also to achieve phagocitosys by AMs. 2.1 Lung deposition To achieve lung deposition, particle size is the most important characteristic to take into account [19]. Figure 4 illustrates the influence of particle size in lung deposition. Particles with diameters greater than 5 μm deposit primarily in the mouth and upper airways, while particles with diameters ranging from 1-5 μm are the most efficient to reach the deep lung. With particles bellow 1µm, mechanisms such as diffusion and sedimentation become important in reaching the pulmonary alveoli, and such could be exploited to optimize pulmonary delivery strategies [20], [21]. Particle size is also 5 Figure 4: Influence of particle size in lung deposition and phagocitosys by AMs. Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs 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 AMs [18]. 2.2 Pulmonary administration Pulmonary administration of drugs must be done using a suitable device. Currently, there are three main delivery devices used for this purpose: nebulisers, pressurized metered-dose inhalers (MDIs), and dry powder inhalers (DPIs). They behave differently and are used with different kinds of particles [22]. MDIs and DPIs are popular choices for the treatment of pulmonary chronic diseases. DPIs are particularly popular, since they are propellant-free, portable, easy to operate and low-cost devices. Unfortunately, dry powders tend to result in particle aggregation, increasing the aerodynamic diameter and lowering the fraction that is respirable, compromising the technique, and rendering them unable to reach the deep lung regions where alveolar macrophages lie. Nebulisers may prove to be a better choice, since they can generally produce liquid droplets which are smaller, and thereby provide the opportunity for a larger proportion of the drug to reach the deep lung regions [17]. Although promising and vastly researched, these delivery strategies face obstacles difficult to overcome. With the particular case of anti-TB drugs, so far not one formulation has reach the market [14]. These difficulties have been reported throughout the scientific literature, and include: the use of safe and accepted excipients, developing scalable processes, developing droplets with proper particle size and morphology for lung deposition, and achieving satisfactory drug loading [14]. Also, usable strategies must be able to account for different lung structures, breathing patterns, and changes in the airway morphology by the pathogenic agent [15]. They must achieve access to poorly-aerated areas of the lung and extracellular bacteria in well-aerated lung tissue, overcoming induction of resistance due to depletion of intracellular drug concentrations, and surpassing limitations due to possible innate responses of the host [23]. The use of nanosystems may be of key interest in overcoming the above-mentioned obstacles. 2.3 Active targeting of alveolar macrophages As stated before, by fine tunning the size of the carrier system, we can enhance phagocitosys by AMs, a desirable event in the case of pulmonary TB. To this passive targeting strategy, there are active targeting strategies that can be used to improve treatment efficacy. In active targeting strategies, 6 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs the constitution and/or structure of the nanosystems is modified, so that certain ligands are present at their surface, changing the way the system interacts with surfaces and cells. Macrophages exhibit a number of receptors that can be exploited by nanocarriers with appropriate ligands. Sugars, such as mannose [24] and lactose [25], are among the most commonly used for this purpose, since these receptors are highly expressed in macrophages. Other ligands commonly used for macrophage targeting include maleylated bovine serum albumin (MBSA), O - steroyl amylopectin (O-SAP), tetrapeptid tuftsin [26], and anionic lipids, such as dicetylphosphate (DCP). 2.4 State of the art Nanotechnology is an area of science regarding the design and study of structures, called nanoparticles (NPs), in which at least one of the dimensions is measured at the nanoscale range (1 nm – 1000 nm). NPs display unique physical and chemical properties that significantly change with their size. In some cases, particles with dimensions greater than 1µm are considered nanoparticles, since they share some, or even most, of these physical and chemical characteristics. NPs can be used for medical purposes, namely as nanocarriers for therapeutic and diagnostic agents by means of encapsulation, covalent attachment, or surface adsorption of these agents [27]. The use of NPs in strategies for pulmonary drug delivery is a promising area of research for several reasons. First, the size of these particles can be fine tuned to reach different areas of the lung, allowing for successful passive targeting strategies. Second, their surface can be modified and ligands attached to actively target bodies of interest, such as AMs [20]. Third, studies have demonstrated that pulmonary delivery of nanosuspensions favor higher lung tissue concentrations and markedly raise the lung to serum ratio of drugs, compared with other routes of administration [28]. This could improve bioavailability, reduce side effects, drug toxicity and dosing frequency, which ultimately leads to the increase of patient compliance and better efficacy of treatment [29]. The most frequent approach in these strategies is the use of neutral nanoparticles as carriers for the drug. Common carriers to achieve pulmonary delivery are lipid NPs, polymeric NPs and lipossomes. Other formulations currently in research include the production of drug nanocrystals, aerosols with magnetic nanoparticles, nanoparticles with effervescent activity, and gold NPs for the study of internalization of NPs by AMs (Figure 5). 7 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs and streptomycin [55]. However, under the tested conditions, rifampicin and ethionamide were not successfully encapsulated. Low encapsulation efficiencies were obtained for isoniazid and pyrazinamide, being the encapsulation of streptomycin only higher at a drug to lipid molar ratio of 0.04. Gaur et al. published a feasibility study where they used rifampicin as the model drug [58]. In this study, in situ formed liposomes showed better sustained release profile than the preformed lipossomes, but both liposomal aerosols showed improved delivery of rifampicin over plain drug aerosols, with encapsulation efficencies around 30%. Liposomes for the delivery of isoniazid have been developed and evaluated in vitro [63] by Chimote and Banerjee. They observed a sustained release of isoniazid encapsulated in liposomes, tooking place over 24 h after a burst release in the first 5h. They have also conducted biocompatibility and stability studies, and found the formulations to be haemocompatible and cytocompatible, and stable for the duration of at least one month. The possibility of surface coating to achieve active targeting with liposomes has also been a subject of interest. Vyas et al. used rifampicin when studying liposomes coated with macrophage-specific ligands, and reported a preferential accumulation of lingad-coated formulations in the lung macrophages, namely MBSA and O-SAP coated liposomes [64]. In vivo tissue distribution studies are on par with these results, by showing higher lung drug concentration for ligand-coated liposomes. O-SAP surface modification was also the focus of Deol and Khuller, who developed coated liposomes for the encapsulation of both rifampicin and isoniazid [65]. They compared the results with uncoated ones, and reported that encapsulating drugs within liposomes reduced toxicity, and that O-SAP coating succeeded in enhancing lung accumulation. Tuftsin functionalization of liposomes encapsulating rifampicin was studied by Agarwal and coworkers [66]. They reported interesting results: considering one single administration, tuftsin functionalization did not give better results than uncoated formulations, but with regular administration over two weeks, tuftsin liposomes were more efficient in controlling tuberculosis. The use of aerosolized liposomes as vaccines to fight TB is a different strategy already considered. Dascher et al. incorporated lipids from MTb into liposomes, and administrated them to guinea pigs [67]. They succeeded in reducing bacterial burden in the lung, but regarding the spleen results were not statistically significant. Moreover, lipid-vaccinated lungs showed significantly less pathology, with granulomatous lesions being smaller and more lymphocytic. Gene therapy has also been the subject of many studies with liposomes for the past twenty years, but despite these efforts, little progress towards developing an effective pharmaceutical product has been done, and the vast majority of clinical trials still uses viral delivery of DNA, a 14 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs much more effective approach, despite the associated toxicity issues [68]. There is ongoing research to address these problems. One recent study used single-tailed cationic lipid 6-lauroxyhexyl lysinate (LHLN) to prepare cationic liposomes, and in vivo results showed that, compared with commercially available Lipofectamine2000/DNA complexes, LHLN-liposomes exhibited lower cytotoxicity, and higher pulmonary gene transfection efficiency [69]. Table 2 summarizes the currently found studies with liposomes as carriers for anti-TB drugs. Table 2: Liposomes for the encapsulation of anti-TB drugs Particle(s) Drug(s) Loading efficiency Size Ligand In vitro/in vivo results Ref. Multilamelar liposomes (PC:Chol) Rifampicin 47 – 49% 2 – 4 µm Negatively charged liposomes (DCP), PE– MBSA conjugate and O-SAP . Lung retention of rifampicin was higher with liposomes than with free drug. The highest value for lung retention was measured in ligand coated liposomes. [64] Pre formed and in situ formed liposomes (EPC:Chol:DCP) Rifampicin 29 – 38% 2 and 1µm for preformed and in situ formed, respectivelly N/A There is no indication of ruptured vesicles for in situ formulations, and prolonged drug release is achieved. In situ formulations do not show any adverse effect on discharge patterns. [58] Liposomes (DSPC:Chol) Isoniazid, pyrazinamide, rifampicin, ethionamide, and streptomycin. 3% for isoniazid, 2% for pyrazinamide, 0% for streptomycin and rifampicin, and 42% for ethionamide (although only at a drug to lipid molar ratio of 0.04). 286 – 329 nm N/A N/A [55] Multilamellar liposomes (DPPC) Isoniazid ~37% 750 nm N/A About 50% liposome entrapped isoniazid released in vitro at the end of 5 h and remaining drug was released slowly over 24 h. [63] Liposomes (PC:Chol) Ciprofloxacin 90% (before nebulization), of which 2 – 30% remained entrapped after nebulization, depending on the nebulizer used. 5 – 7 µm N/A N/A [59] 15 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs Table 2: Liposomes for the encapsulation of anti-TB drugs Particle(s) Drug(s) Loading efficiency Size Ligand In vitro/in vivo results Ref. Liposomes (DPPC:Chol and EPC:Chol) Ciprofloxacin 97% before lyophilization, of which up to 90% were retained by the lyophilized cake, and up to 40% after jet milling. 1 – 7µm (lyophilized cake); 1 – 2 µm (after jet milling) N/A N/A [60] Liposomes (DPPC:CHOL: DSPE-MPEG and DPPC:CHOL:DS PE-PEGNH2) Ciprofloxacin N/A 140 – 460 nm for liposomes, depending on formulation. 1 – 140 µm for agglomerates (AVT1 and AVT2). N/A In vitro studies showed liposomes, AVT 1, and AVT 2 had an initial burst in release of drug, but it was much lower than that for free ciprofloxacin, and were capable of extended drug release in the blood. After instillation of cysteine at 90 minutes into the lungs of the rabbits treated with AVT 2, an elevation in release rate was observed. [62] Liposomes (DSPC:Chol) MTb wholelipid extract incorporated in the liposomes N/A < 300 nm N/A Lipid-immunized animals showed reduced bacterial load in the lung, but no statistically significant decreases in the spleen. Lesions in the lung tissue of lipidand BCG-vaccinated animals were smaller, less necrotic and more lymphocytic. [67] Liposomes (DMPG, EPC:DMPG, and DMPC:DMPG) Ciprofloxacin Above 90% and around 50%, before and after nebulization, respectively. 2 – 3 µm N/A N/A [61] 16 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs Table 2: Liposomes for the encapsulation of anti-TB drugs Particle(s) Drug(s) Loading efficiency Size Ligand In vitro/in vivo results Ref. Liposomes (EPC:Chol) Isoniazid, rifampicin 8 – 10% for isoniazid. 44 – 49% for rifampicin. ≥200 nm for O-SAP coated liposomes. <200nm for DSPE-PEG liposomes. O-SAP Encapuslated drugs were found to be less toxic than free drug. Drug uptake in macrophages was found to be similar between encapsulated and free drugs. Slow and controlled drug release was achieved in encapsulated drugs. O-SAP coating enhanced lung accumulation. Also, pre-administration of PC and Chol liposomes before the injection of lung specific stealth liposomes, further enhanced their uptake in lungs. [65] Liposomes (EPC) Rifampicin. 28 – 32% 25 – 65nm Tuftsin With 10 mg/kg dose of liposomal RIF, a significant reduction in the lung bacillus load and an increase in MST were observed, compared with those in free RIF treated animals. Regarding tuftsin functionalization, one single treatment with coated liposomes was only marginally better than that observed with uncoated ones, but coated liposomes given twice weekly for 2 weeks was considerably more effective than uncoated ones in controlling TB. [66] 2.4.3 Drug nanocrystals The pure use of therapeutic agents in the form of nanocrystals has been proposed as a system for drug delivery. They are used as dispersions of pure drug nanoparticles kept stable through the presence of a minimum amount of a surfactant – nanosuspensions. Drug nanocrystals dissolve rapidly in the lung lining fluid leading to a high concentration, which is helpful for localized treatment of respiratory diseases such as pulmonary TB. Results show that these could be used in drug delivery formulations to improve pharmacokinetic, pharmacodynamic and targeting properties of poorly soluble drugs. Gao et al. reported two different kinds of pulmonary formulations containing drug nanocrystals [28]: aqueous nanosuspension packaged and administered by a nebulizer; drug nanocrystals collected and transported into the lung by the small aerosol droplets generated by the nebulizer. 17 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs Spore like drug particles for deep lung deposition have also been proposed as an innovative system [70]. Hollow and spore like nanoagglomerates were obtained by mixing the drug solution with an antisolvent in a high gravity environment. The fabrication of drug particles similar to spores may improve the pulmonary drug delivery efficiency in DPIs, and is a more efficient, cost-effective and easy to scale up method over milling, homogenization, spray freezing into liquid, and supercritical antisolvent precipitation to prepare nanosuspensions. According to the authors, uniform particle size and controlled morphology can be achieved with this technique. Currently, only one report was found regarding the production of nanocrystals or nanoagglomerates of an anti-TB drug. El-Gendy et al. prepared ciprofloxacin nanosuspensions that were then flocculated to form nanoparticle agglomerates [71]. Nanoparticle size ranged from 68 – 722 nm, depending on the formulation, and agglomerates exhibited a particle size range of 2 – 4 μm. They performed dissolution studies, and compared the results with the stock drug. Results showed that the dissolution rate was improved, demonstrating that these techniques may help to overcome some of the solubility issues presented by new anti-TB drugs, specially by molecules that, although did not pass from the clinical trials due to solubility issues, shown higher potential as anti-TB drugs. 2.4.4 NPs with effervescent activity Nanoparticles with effervescent activity have recently been suggested for pulmonary delivery. Oral drug delivery associated with effervescent pharmaceutical formulations is used for a long time, in stomach distress medications, vitamin supplements and analgesics. Effervescent activity of the carrier particles occurs when the carrier particles are exposed to humidity, adding an active release mechanism to the pulmonary route of administration. Additionally, effervescent particles can be synthesized with adequate size for deep lung deposition, and the technology appears to be safe for pulmonary delivery [72]. Although effervescent NPs have been mostly studied as a promising pulmonary delivery strategy for anti-cancer drugs [72]–[74], one report has been found regarding their use for the delivery of ciprofloxacin [75]. Ely and coworkers have developed and studied different powder compositions with effervescent activity, and found two formulations suitable for pulmonary delivery. These formulations had the addition of l-leucine and PEG 6000, which improved the aerodynamic characteristics of the powder particles. Effervescent activity of the prepared formulations resulted in the release of nanoparticles with less agglomeration compared to the carrier particles made just of lactose. 18 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs Table 3: Other nanosystems for the delivery of anti-TB drugs Particle(s) Drug(s) Loading efficiency Size Ligand In vitro/in vivo results Ref. Drug nanoparticle agglomerates Ciprofloxacin 81 – 96% 68 – 722 nm for NPs; 2 – 4 μm for NP agglomerates N/A N/A [71] PBCA Effervescent NPs Ciprofloxacin N/A Carrier particles: 2µm Effervescent preparations: 244 and 252 nm before and after spray drying, respectively. Effervescent preparations containing lleucine and PEG 6000: 150 and 177 nm, before and after spray drying, respectively. N/A Effervescent carrier particles released 56% ciprofloxacin into solution compared with 32% when lactose particles were used. [75] 2.4.5 Gold and magnetic NPs Gold NPs have recently been used to study internalization and intracellular translocation of inhaled nanoparticles in rat AMs [76]. Particles used had mean hydrodynamic radius of 16 nm. Results showed AMs had efficiently internalized NPs by endocytosis. Gold NPs have been conjugated with streptomycin [77], and it has been demonstrated that ciprofloxacin binds to gold NPs [78], but the cited studies do not focus on pulmonary delivery, and no other reports have been found regarding the use of these particles for pulmonary delivery, regardless of the model drug. The use of magnetic aerosols using superparamagnetic iron oxide NPs has also been suggested as a way to improve drug delivery to the lung [79], and ciprofloxacin has been used as a model drug in the development of superparamagnetic nanocomposites with magnetically mediated release of the loaded anti-TB drug [80]. However, no study has been found combining these two strategies to achieve magnetically mediated pulmonary delivery of anti-TB drugs. 19 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs 2.4.6 Lipid NPs Lipid NPs are the last nanosystems presentd in this state of the art and are the focus of this thesis. Generally speaking, and by contrast with liposomes and polymeric nanoparticles, lipid NPs show higher drug loading capacity, higher stability, and require the use of lower amounts of organic solvents during production [81]. As with liposomes and most polymeric NPs, these nanocarriers are biocompatible and can be produced with appropriate size and morphology for lung targeting and deposition [82], and have been studied as a viable pulmonary drug delivery strategy [83]. It is also possible to modify the surface of lipid NPs to achieve active targeting of AMs. Mannose is a common surface modification with lipid nanocarriers [84]. Solid lipid nanocarriers (SLNs) and nanostructured lipid carriers (NLCs) are the two most common lipid NPs used. The published results by Jain and coworkers, who compared four different nanocarriers for the incorporation of ciprofloxacin, showed that SLNs are capable of prolonged drug release [38]. This work is one of the three reports that were found regarding pulmonary delivery of SLNs loaded with drugs for the treatment of TB, namely rifabutin, isoniazid, rifampicin and pyrazinamide. Nimje et al. prepared rifabutin loaded SLNs, and compared uncoated formulations with formulations coated with mannose [24]. Results showed cellular uptake in AMs was almost six times enhanced due to mannose coating. Coated formulations also showed to be less immunogenic and more suitable for sustained delivery. Pandey and Kuller have prepared SLNs for pulmonary delivery through nebulization [85]. They incorporated isoniazid, rifampicin and pyrazinamid, of which rifampicin showed the highest incorporation due to the lipid-based nature of the formulation and lipophilic characteristics of the drug. The nebulized SLNs were successfully deposited in the lungs, and were detected in other organs up to 7 days after administration. Administrated free drug was cleared from the system within 24 – 48 h. Jain and Banerjee included SLNs in their list of nanosystems to deliver ciprofloxacin, and concluded that these NPs were suitable for drug loading, and capable of sustained drug release [38]. 20 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs The matrix of SLNs consists of solid lipids only, with perfect crystallinity. This results in lower drug loading, since there are very few empty spaces in which the drug can be found. It also results in expulsion of drug content during long storage due to changes in lipid packaging. NLCs are different structures. The matrix consists of both solid and liquid lipids, consequently showing lower crystallinity and higher incidence of nanostructures, which won't result in denser lipid packaging over time. Thus, higher drug loading and stability during long storage is achieved, when compared with SLNs [86]. Figure 6 illustrates the differences in the matrix of SLNs and NLCs, and the overall influence of such differences in drug loading and expulsion over time. However, it should be noted that no study was found regarding the use of NLCs as carriers for any anti-TB drug. Table 4 summarizes currently found studies regarding the use of lipid nanoparticles for the treatment of TB. 21 Figure 6: Schematic representation of the matrix of SLN and NLC. A:SLN exhibits a high order matrix, while NLC exhibits a low order matrix. B: Drug is loaded into SLNs and NLCs. C: Over time, SLNs tend to a denser lipid packaging and higher drug expulsion than NLCs. Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs Table 4: Lipid NPs for the incorporation of anti-TB drugs Particle(s) Drug(s) Loading efficiency Size Ligand In vitro/in vivo results Ref. SLN (tristearin) Rifabutin 82% (uncoated) and 87% (coated) 251 nm (uncoated) and 389 nm (coated) Mannose In vitro studies showed a sustained drug release for 120h, during which uncoated SLNs showed higher drug release. Macrophage uptake was higher for coated SLNs. In vivo results showed higher drug presence for coated SLNs in the lungs. [24] SLN (Stearic acid) Isoniazid, rifampicin and pyrazinamid 51% for rifampicin, 45% for isoniazid and 41% for pyrazinamide. 1 – 2 µm N/A In vitro results varied for simulated gastric or intestinal fluid. The drug released was <20% in the first 6 h and 11– 15% during 6–72 h for isoniazid/pyrazinamid; 9% in the first 6 h and 11% during 6– 72 h for rifampicin, although rifampicin release was in the range of 8–12% during the entire study period for intestinal fluid. All the three drugs could be detected in the lungs, liver and spleen of the animals up to day 7 following the nebulization. [85] SLN (stearic acid) Ciprofloxacin 39% 74 – 99 nm N/A The aim of this study was to compare four different nanosystems: SLNs, albumin, gelatin and chitosan. In vitro results showed SLNs were capable of a prolonged drug release up to 80 h. [38] 22 3 Materials and Methods 3.1 Development of Nanostructured Lipid Carriers 3.1.1 Initial formulation NLCs were initially prepared with with Cetyl Palmitate (C32H64O2; M=480.83 g mole-1; Gattefossé) as the solid lipid, and Mygliol 812 (from Acofarma) as the liquid lipid. Polysorbate 60 (C64H126O26; M=1 310 g mol-1; Sigma Aldrich) was used as surfactant, to stabilize the emulsion. Quantities used for each are presented in table 5: There are several methods to produce NLCs in the laboratory, such as high pressure homogenization, microemulsion technique, emulsification-solvent evaporation, emulsification-solvent diffusion method, solvent injection (or solvent displacement) method, phase inversion, multiple emulsion technique, ultrasonication and membrane contractor technique [86]. In the present work, an ultra-sonication method was used. In detail, the two lipids and the surfactant were heated in a water bath up to 70º C, temperature at which both lipids are in the liquid state. When the solid lipid was fully melted, 4.4 mL of heated ultrapure water (T = 70º C) was added to the mixture. Mixture then went through ultraturrax (Ystral X10/20 E3) at 3500 rpm for 30 s, and sonication (Sonics Vibra-cell, with CV18 probe) at 70% power for 5 min, which resulted in a nanoemulsion. This nanoemulsion was finally left to cool at room temperature and stored. Figure 7 features a schematic representation of the process. 23 Mass / mg Cetyl palmitate 350 Mygliol 812 150 Polysorbate 60 100 Formulations prepared with 4.4 g of ultrapure water. Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs generate an electric field, which results in a movement that overlaps Brownian motion. The frequency in scattered light thus shifts due to Doppler effect (Figure 11), and that shift (Δω) can be used to calculate particle drift velocity, through the equation: Δ ω=q.vd (4) where q is the scattering vector already discussed in the previous section. The magnitude of drift velocity is proportional to the electric field (E) by a constant, μ, which is defined as the electrophoretic mobility of the particle: vd=μ×E (5) Finally, the ζ-potential can be calculated from the electrophoretic mobility, taking electrokinetic properties of the medium into account, through mathematical models. One of these models, and the one used in this work, is the Smoluchowski equation: ζ=ημ ε0ε (6) where η is the viscosity of the medium, ε0 is the permitivity of vauum, and ε is the dielectric constant of the medium [97]. ζ-potential of NLCs was measured using a BI-MAS DLS instrument (Brookhaven Instruments, USA), operating at scattering angle of 90°, a temperature of 25 °C, pH 7.4. For each measurement, 10 runs were performed. 3.2.3 Particle morphology Scanning Electron Microscopy (SEM) was used to observe particle morphology. In electron microscopy, a beam of charged particles (electrons) is used instead of a beam of photons (used with 30 Figure 11: Frequency shift of scattered light due to movement of suspended particles when subjected to an electric field. Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs optical microscopy). These particles provide beams with high energy, thus shorter wavelengths, hence allowing an observation of objects in the nanometric scale. In SEM, this beam interacts with small areas of the sample, and secondary electrons (among other particles) are scattered from it. These electrons are detected and the beam proceeds in scanning the rest of the surface. Gathered data can then be used to reconstruct the sample's morphology. The signals obtained with SEM also allow for the determination of chemical composition, crystalline structure and orientation of materials making up the sample [98]. For the present work, the SEM exam was performed using a high resolution Scanning Electron Microscope with X-Ray Microanalysis and CryoSEM experimental facilities: JEOL JSM 6301F/ Oxford INCA Energy 350/ Gatan Alto 2500. The specimen was rapidly cooled (plunging it into subcooled nitrogen – slush nitrogen) and transferred under vacuum to the cold stage of the preparation chamber. The specimen was fractured, sublimated (‘etched’) for 120 sec. At –90 °C, and coated with Au/Pd by sputtering for 45 sec. The sample was then transferred into the SEM chamber, and studied at a temperature of -150 °C. 3.2.4 Loading efficiency The efficiency with which an active agent is incorporated into a carrier matrix can be expressed as a percentage, which is referred to as the loading efficiency (LE), and is calculated using the following expression: LE=TD−UD TD ×100 (7) where TD is total drug and UD is unentrapped drug (drug dissolved in water medium). The loading capacity (LC), which is the capacity of an encapsulation system to carry a drug, can be calculated by the expression: LC=ED ED+matrix ×100 (8) where ED is the drug entrapped in the nanosystem. The amount of drug dissolved in water medium can be quantified by UV-Visible spectroscopy, which allows relating absorbance of radiation at a specific wavelength with the concentration of the dissolved drug. 31 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs When electromagnetic radiation interacts with matter, energy transfer may occur in many ways. In one of them, electrons in the electron cloud will absorb incident photons, as long as they have the exact same energy as it would be necessary to promote the transition from one energy level to an upper one, changing the atom or molecule's quantum mechanical states. The allowed energy levels are quantized, and are specific of the chemical species [99]. The energy of an electromagnetic wave, E, is related to it's frequency, f, by the expression: E=h.f=c h λ (9) where h is Planck's constant. Since f=c/λ, where c is the speed of light in vacuum and λ is the wavelength of radiation. It is therefore possible to identify elements in samples by determining for which wavelengths photons are highly absorbed from an incident beam. This set of wavelengths constitutes an absorption spectrum [100]. Absorbance of a solution is proportional to the solute's concentration, as is expressed by Beer-Lambert's law: A=abc (10) where c is the solute's concentration, b is the length of solution the beam has to cross, and a is a proportionality constant. A calibration curve is usually obtained by preparing several samples, with increasing, well known concentrations, and obtaining their absorption spectrum. Prominent local maximums, indicating high absorbance at specific wavelengths, are identified. For the wavelength at a specific local maximum, and ab being constant, absorbance is plotted against concentration, and a linear fit should be obtained. The resulting equation is then used to determine the mass concentration of a sample, through measuring the absorbance at that particular wavelength, provided the expected concentration is inside the range used for calibration [100]. A modern spectrophotometer does an automatic and highly precise wavelength sweep, meaning it automatically changes the wavelength of the incident beam by controlling a monochromer. The first step in the determination of incorporated drug, was to obtain a calibration curve from drug solutions with known concentrations. The determination of the concentration of the two drugs used in the present work was done with a spectrophotometer (Jasco V-660) using quartz cells. Wavelength interval ranged from 200 nm to 700 nm, with 1.0 nm intervals. The prepared concentrations of drug to determine the linear fit are presented in table 8 for RIF and in table 9 for RFB. The second step consisted in separating the NLCs from their supernate, and then measuring the drug content of the supernate (non-loaded drug). To achieve this, formulations were diluted and 32 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs centrifuged inside centrifugal filter units (50 000 NMWL, Milipore corporation, Ireland) for 5 min at 3 500 RPM. Supernate was collected and it's drug content determined as explained before. 3.2.5 Schiff's base detection by FTIR spectroscopy As stated before, mannose coating was done by bonding of the open chain mannose with amine terminations in the surface of the NLCs through a Schiff's base (see section 3.1.4). Fourier Transform Infrared (FTIR) spectroscopy was used to detect Shiff's base and confirm coating with mannose. FTIR spectroscopy is a technique similar to UV-Vis spectroscopy. The main differences between the two techniques reside in the wavelength of the incident beam and in the mathematical treatment of the raw data. UV-Vis spectroscopy works in the ultraviolet and visible ranges of the electromagnetic spectrum, while FTIR, as the name suggests, works in the infrared range. The difference in wavelength translates in a difference in energy, and FTIR spectroscopy is therefore able to detect energies associated with specific bonds inside molecules. The second mentioned difference is related with how the data is obtained. In UV-Vis spectroscopy, beams of very well defined wavelengths are used for each measurement. With FTIR spectroscopy, broadband beams, containing full spectrum of wavelengths, are used for each measurement. Following measurements use different spectra. The translation of raw data into absorption for each wavelength requires mathematical processing, and a Fourier transform is used. The fact that a full spectrum of wavelengths is used instead of a single wavelength beam, renders this technique faster than conventional spectroscopy. FTIR analysis of the samples was done with a Frontier FTIR Spectrometer with universal ATR Diamond/ZnSe accessory (Perkin Elmer, USA). Samples were lyophilized as described in section 3.1.5 before FTIR analysis. Lyophilized samples were placed in ATR accessory and samples were measured from wavenumber 600 to 4 000 cm-1, 50 scans per sample, with a resolution of 4 cm-1. 3.2.6 Citotoxicity Citotoxicity of the developed formulations was studied using MTT (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide) assays with three different cell lines: Calu-3 (upper respiratory tract epithelium); A529 (lower respiratory tract epithelium ) and Raw (macrophage cell lines). MTT is a water soluble salt. Cleavage of the tetrazolium ring of MTT by succinate dehydrogenase within the mitochondria results in the formation of an insoluble purple dye, which is impermeable to cell membranes. For this reason, the dye accumulates in the interior of non damaged cells. This insoluble dye can be collected and dissolved, resulting in a colored solution. Through the measurement 33 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs of the absorption of light of this solution at a specific wavelength through spectroscopy, it becomes possible to determine the amount of cells killed by a given process [101]. MTT assay was performed in the course of three days. In the first day cells were seeded to the 60 interior wells of a 96 well plate (the 36 exterior wells were filled with PBS), through the following procedure. Cells were cultivated in T25 flasks with Dulbecco's Modified Eagle Medium (DMEM) + Glutamax, complemented with PenStrep, Fungizone and Fetal Bovine Serum (all from Gibco, Life Technologies). When confluency was reached, cell medium was removed, and flasks were washed two times with phosphate buffer saline (PBS) solution, and medium was added. Cells were then detached from the flask's surface. For Raw cell lines, this was done using a scraper. To detach Calu-3 and A549 cells, a tripcine solution was added and the flasks were put to rest for 5 min at 37 ºC. Medium with suspended cells was pipetted to falcon tubes, that were then centrifuged (1 500 rpm, 5 min, 4 ºC) to separate medium with tripcine from the cell's pellet. Cells were then resuspended in DMEM. All cells were then counted using the tripanblue explosion method, with a Neubauer chamber, and cell suspensions were diluted to obtain concentrations of 25 000 cells mL-1. Finally, each suspension was added to each of the 60 wells, to achieve an approximate concentration of 5 000 cells/well. Cell plates were left to rest for 24 hours, at 37 ºC. In the second day, medium from wells was removed and cells were washed 2 times with PBS. NLC suspensions and positive and negative controls were added to the wells. Positive control was DMEM. Negative control was Triton X100 2% (m/m). Lyophilized NLCs were resuspended in DMEM. For each NLC formulation, 5 concentrations were used (1 µg mL-1, 10 µg mL-1, 100 µg mL-1, 1 000 µg mL-1 10 000 µg mL-1), and for each concentration 5 wells were used. Cell plates were left to rest for 24 hours, at 37 ºC. In the third day, MTT was added to the wells and cell plates were left to rest for 4 hours. At the end of this period, medium was removed, and Dimethyl sulfoxide (C2H6OS, M = 78.13 g mol-1, Sigma Aldrich) was added to each well. Cell plates were protected from light and agitated for 5 minutes. Then absorptivity was measured using a multi-mode microplate reader (Synergy HT, Biotek Instruments). This three day procedure was repeated two times for each cell line to replicate results. 34 4 Results and discussion 4.1 Particle size and Zeta potential Mean hydrodynamic particle size for all formulations was found to be around 200 nm, as it is possible to see in tables 6 and 7. PDI was found to be above 0.100 in all formulations. This indicates that none of the formulations are monodisperse. As stated in section 3.2.1, prior to size and zeta measurements, formulations were filtered with a syringe filter with a porus of 800 nm. The objective of this step was to remove any excess lipid and particle agglomerates. However, it does allow particles of bigger sizes to coexist in the formulation if the method produces them. The conclusions to be taken from these results are that the method used in this work does not produce monodisperse NLC formulations without posterior filtering, but the obtained sizes are within the breathable range, and with potential to reach the pulmonary alveoli. Table 6: Mean hydrodynamic particle size and zeta potential for unloaded formulations NLC NLC-M Mean size / nm 174.4 ± 2.4 197.4 ± 1.4 PDI 0.211 ± 0.012 0.120 ± 0.008 Zeta potential / mV – 20.5 ± 0.8 48.7 ± 2.1 Results: mean ± SD (n ≥ 3) Results for ζ – potential listed in table 6 show that NLC have a pronounced negative charge, while NLC-M exhibit a high positive charge. These values point to a successful mannosylation of the NLCs. As described in section 3.1.4, the process of mannosylation involves adding stearyl ammine to the formulations, so that nanoparticles have amine groups at the surface. These amine groups are expected to link to mannose molecules through a Schiff's base. Amine groups are positively charged, so ζ – potential was expected to become positive. These results are also observed in table 7, with drug loaded NLCs. All mannosylated formulations exhibit positive charges, contrasting with non-mannosylated formulations. This indicates us that the presence of RIF and RFB in the formulations does not interfere with the mannosylation process. 35 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs Table 7: Mean hydrodynamic particle size and zeta potential for loaded formulations. NLC-RIF NLC-M-RIF NLC-RFB NLC-M-RFB Mean size / nm 210.6 ± 1.2 202.0 ± 3.5 179.1 ± 1.6 213.0 ± 1.8 PDI 0.129 ± 0.007 0.190 ± 0.008 0.114 ± 0.015 0.118 ± 0.014 Zeta potential / mV -20.8 ± 0.9 31.9 ± 0.5 -17.9 ± 1.9 37.6 ± 1.0 Results: mean±SD (n ≥ 3) Surface charge plays an important role in particle stability. The observed absolute values for ζ – potential are high, both for NLC and NLC-M. This indicates that the formulations are presumably stable. Stability of two formulations was monitored through time. Size and ζ – potential was measured at day 1, day 30, day 90 and day 180. Results up to day 90 are shown in figure 12 to 14. Mean size and PDI are shown for NLC (figure 1) and NLC-M (figure 2), and they do increase over time. Figure 12: Mean size and PDI (± SD) of NLC. Figure 13: Mean size and PDI (± SD) of NLC-M. Figure 14 shows a slight shift of ζ – potential torwards neutrality. However, more dramatic were the observations at day 180. These show formulations with visible particulates in suspension. DLS analysis was not possible, as the software was not able to determine a correlation function from the acquired data. This points to a shelf stability of the formulations inferior to 6 months. 36 0 20 40 60 80 100 160 180 200 220 0.10 0.15 0.20 0.25 SIZE PDI Days Mean Size / nm PDI Figure 14: ζ - potential ± SD for NLC and NLC-M 20 40 60 80 100 -40 -20 0 20 40 60 NLC NLC-M DAYS ZETA Potential / mV 0 20 40 60 80 100 160 180 200 220 240 260 0.15 0.20 0.25 0.30 SIZE PDI Days Mean Size / nm PDI Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs 4.2 Particle morphology SEM imaging revealed spherical particles for all formulations, with sizes between 100 and 300 nm. Both these observations validate the results obtained with DLS, and the method of formulation. As we can see in figures 15A to 15B, it can be observed that both NLC and NLC-M formulations exhibit similar morphologies, indicating that the method of mannosylation does not have an effect on particle shape. However, images for NLC-M do show smaller particles than NLC. A possible explanation is that such is simply due to the polidispersity. Different regions may exhibit differences in observed sizes. To test this hypothesis, a greater number of images should be obtained, particles should be individually counted and measured, and the results should be compared with the results from DLS. NLC A B NLC-M CD Figure 15: SEM images for NLC and NLC-M 37 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs From figures 16A to 16B, it can observed that loaded NLCs (NLC-RIF and NLC-RFB) also exhibit spherical shapes, which indicates that the method of drug loading is also not resulting in morphological changes. The variability of sizes seems to be higher in drug loaded NLCs (this is more visible in NLC-RIF images) than with empty NLCs, but this was not observed in PDI measured with DLS. Again, the region of observation plays an important role in the perception of the characteristics of each sample, reinforcing the complementary nature of the different methods used to evaluate particles size and PDI. NLC-RIF AB NLC-RFB C D Figure 16: SEM images for NLC-RIF and NLC-RFB 38 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs 4.3 Loading efficiency Figures 17 and 18 show spectrograms for the absorbance obtained by NLC-RIF and NLC-RFB, respectively. For RIF, spectrum showed three interesting local maximums at 255, 334 and 473 nm. Linear fit of the plot of concentration versus absorbance was obtained, and wavelength for 334 nm showed greater R² value. For RFB, spectrogram showed interesting local maximums at 208, 279 and 319 nm. Linear fit of the plot of concentration versus absorbance resulted in wavelength 208 nm showing greater R² value. 39 Figure 17: Calibration spectrum and linear fit for RIF. 200 300 400 500 600 700 0 1 2 3 4 Absorbance Wavelength (nm) A B C D E F G H 255 334 473 0 0,02 0,04 0,06 0,08 0,1 0,12 0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 f(x) = 20,4284757953x - 0,0306470904 R² = 0,9982702003 f(x) = 31,2535731794x - 0,0019175186 R² = 0,9991135641 f(x) = 38,0909892556x + 0,0086881191 R² = 0,9958098287 255 Linear (255) 334 Linear (334) 473 Linear (473) Cm / mg mL ¹⁻ Absorbance Table 8: Concentrations of RIF solutions used in dosing calibration. Solutions A B C D E F G H Cm / mg mL ¹⁻0.1000 0.0500 0.0278 0.0167 0.0100 0.0050 0.0028 0.0006 Development and Characterization of Nanocarrier Systems for the Delivery of Antitubercular Drugs Precirol ATO 5 was chosen for the final formulation. This resulted in LE that ranged from 82.4 ± 5.7 % to 87.5 ± 4.6 %, for a drug to lipid ratio of approximately 1:70. Cytotoxicity of the NLCs was studied by MTT assay, using RAW, CALU-3 and A549 cell lines. Results showed that it was possible to reach concentrations above 100 and below 1 000 μg mL-1 before IC50 was reached, for drug loaded formulations. Other toxicity studies, such as the lactate dehydrogenase assay, could be used in the future to complement these results. Other in vitro studies may shed a light on the real possibilities of these nanocarriers. Cellular uptake studies should be performed to access if the use of NLCs does enhance the uptake of drugs, and if mannose coating indeed improves these values. After all in vitro studies are duly performed, in vivo studies should be considered. These studies should be planned to answer some pertinent questions. 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