Oxaprozin-loaded lipid nanoparticles towards overcoming NSAIDs side-effects

OXAPROZIN-LOADED LIPID NANOPARTICLES TOWARDS OVERCOMING NSAIDS SIDE-EFFECTS JOSÉ PLÁCIDO LOPES DE ARAÚJO DISSERTAÇÃO DE MESTRADO APRESENTADA À FACULDADE DE ENGENHARIA DA UNIVERSIDADE DO PORTO EM BIOENGENHARIA – BIOTECNOLOGIA MOLECULAR M 2014 © José Araújo, 2014 Faculdade de Engenharia da Universidade do Porto Instituto de Ciências Biomédicas Abel Salazar Oxaprozin-loaded Lipid Nanoparticles towards overcoming NSAIDs side-effects José Plácido Lopes de Araújo Master Thesis for the degree in Master of Science in BioEngineering – Specialization in Molecular Biotechnology Supervisors: Prof. Dr. Salette Reis Dr. Cláudia Nunes July 2014 ii © José Araújo, 2014 i Abstract The inflammatory process is the innate immune response for the presence of pathogens, toxic molecules, tissue injuries or any other harmful conditions. The inflammation process is characterized for redness, pain, swelling, heat and disturbance of function and comprises inducers, sensors, mediators and effectors components from cellular and humoral origin. Macrophages are one of the most important cells in the inflammatory process. Macrophages actively phagocyte particles with sizes superiors to 200 nm and express folate receptor making them of great interest for passive and active targeting strategies. Non-Steroidal AntiInflammatory Drugs, like oxaprozin, are one of the most used drugs prescribed for these conditions, however these drugs have adverse side effects, namely at the level of the gastric mucosa, that must be avoided and pharmacokinetic properties that need to be improved and for these purpose many delivery systems arise. Lipid Nanoparticles allow an effective drug packaging and targeted delivery, improving drug´s pharmacokinetics and pharmacodynamics properties and avoiding some of their side effects. In this work, two formulations containing oxaprozin were developed: nanostructured lipid carriers with and without folate functionalization. Folate functionalization was obtained by the addition of a synthesised DSPE-PEG2000-FA conjugate. These formulations had low polydispersity (approximately 0.150) with mean diameters around 285 nm and zeta potential superior to -40 mV. The elevated encapsulation efficiency of the particles (superior to 95%) along with the high amount of used oxaprozin lead to a high loading capacity (close to 9%). The formulations avoided the oxaprozin release in simulated gastric fluid promoting its release on simulated intestinal fluid, physiologic and inflammatory medium, remaining only less than 10% of the oxaprozin entrapped on the lipid carrier matrix. MTT and LSH assays revealed that the formulations only seemed to present cytotoxicity in Caco-2 cells for oxaprozin concentrations superiors to 100 μM. Permeability studies in Caco-2 cell lines shown that oxaprozin encapsulation did not interfere with oxaprozin permeability, having the final formulations an apparent permeability around 0.8x10-5 cm.s-1 in simulated intestinal fluid and about 1.45 x10-5 cm.s-1 PBS. Keywords— Inflammation, Oxaprozin, Nanostructured Lipid Carriers, Nanoparticles Characterization, Folate Functionalization, In vitro Release Study, MTT assay, LDH assay, Caco-2 Permeability study. ii -This page was intentionally left blank- iii Acknowledgments I express my deepest and sincere gratitude to Professor Salette Reis for once again giving me the opportunity to work with his research group at the Faculty of Pharmacy and for hall the guidance, encouragement and concern. I am deeply grateful to Dr. Cláudia Nunes for these two years of guidance and care! I am also deeply sorry for being so annoying sometimes, but that was my “job”. I want you to know that if you are the guilty for all the things that go wrong on the lab and all the “open doors” you also are the guilty for everything that had gone well, for everything that I have learn and all the doors that may have been open to my areas of knowledge and who knows my future. I would also like to thank all the girls and couple of boys of the lab for all the good moments, help and kind words along the time and for making the job more fun and relaxed. I let a special thank for Nini and Virgínia for the special help and work on some experimental points. To my friends “Tecos” for the moments of support, all the friendship, talks, laughs and “milkshake” moments with no sense at all but with all the sense in the world. But that is how we are... To my parents, brother and grandmother, without you this work have been impossible. Thank you for all the support, education, values and the effort that you have made for making my dreams come true. And I want to thank Daniela for being always there with her unconditional and endless love, continuous support and encouraging smile! iv -This page was intentionally left blank- v Table of Contents Abstract ............................................................................................i Acknowledgments ............................................................................. iii Table of Contents ............................................................................... v List of Figures ................................................................................. vii List of Tables ................................................................................. viii Glossary .......................................................................................... ix Chapter 1 ......................................................................................... 1 Introduction ....................................................................................................... 1 1.1 - The inflammatory process ............................................................................ 1 1.2 - Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) ................................................ 3 1.3 - NSAIDs delivery systems .............................................................................. 7 1.4 - Passive and Active Targeting ...................................................................... 10 1.5 Lipid Nanoparticles (LNs) ........................................................................... 12 1.6 Oral Administration .................................................................................. 15 1.7 Specific aims and strategy ......................................................................... 16 Chapter 2 ....................................................................................... 17 Materials and Methods ........................................................................................ 17 2.1 - Materials ............................................................................................... 17 2.2 - Methods ................................................................................................ 17 Chapter 3 ....................................................................................... 25 Results and Discussion ........................................................................................ 25 3.1 - Choice of solid lipid for improved drug loading ................................................ 25 3.2 - Lipid nanoparticles synthesis and optimization ................................................ 26 3.3 - Nanoparticles characterization .................................................................... 36 3.4 - In vitro release studies ............................................................................. 39 3.5 - Effect of LNs on cell viability and cytotoxicity ................................................. 41 3.6 - Cell permeability studies ........................................................................... 44 Chapter 4 ....................................................................................... 47 Conclusions...................................................................................................... 47 1 Chapter 1 Introduction Chronic inflammation conditions are on the base of several diseases, reason why the development of effective therapies in the treatment of these conditions is of extreme importance [2-5]. Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are one of the most used drugs prescribed for these conditions however, like every drug they present adverse side effects [6-9]. In order to surpass these adverse effects, specialized delivery systems may be of extreme importance and may hold the answer for an effective anti-inflammatory treatment of such conditions. 1.1 - The inflammatory process The presence of pathogens, toxic molecules, tissue injuries or any other harmful conditions in an organism will origin a defensive response in order to restore the cellular homeostasis [2]. The human body comprise three distinct defence systems that include the anatomic and physiologic barriers, the innate immunity and the adaptative immunity [10]. The innate immune response is characterized by an inflammatory process that is traditionally defined by four symptoms related to the immune system’s response. These wellknown symptoms are redness, pain, swelling and heat (in Latin rubor, dolor, tumor, and calor respectively) [2, 3, 5, 11, 12]. Another symptom is disturbance of function (in Latin functio laesa), which was posteriorly added to the previous four [3, 5, 12]. These symptoms have origin in vascular and cellular changes promoted by inflammatory mediators that are produced by inflammatory and tissue cells or by metabolization of their extracellular precursors [12]. The inflammatory process includes cellular elements: monocytes, macrophages, neutrophils, dendritic cells, and natural killer cells (whose main functions are to phagocyte and kill the inflammatory agents and antigen-presentation), eosinophils, basophils and mast cells (responsible for the production of the inflammatory mediators) and humoral elements: complement, lysozime, lactoferrin and antimicrobial peptides [5, 10, 11, 13, 14]. The components of the inflammatory process can be characterized as inducers, sensors, mediators and effectors and this process is summarized in the Figure 1. The origin of the inducers determines their classification. Exogenous inducers can be divided in microbial inducers: Pathogen-Associated Molecular Patterns (PAMPs) and virulence factors; and non-microbial inducers which include toxic compounds, foreign bodies, irritants and allergens. Endogenous inducers are generally signals produced by tissues in response to stress, damage or malfunctioning, designated Danger-Associated Molecular Patterns (DAMPs) [2, 14]. 2 Sensor components comprise specific membrane receptors like Pattern-Recognition Receptors (PPRs) that recognise PAMPs and DAMPs, Toll-Like Receptors (TLRs), C-type Lectin Receptors (CLRs), NOD-like Receptors (NLRs), Mannose Binding Protein (MBP) and Lipopolysaccharide Binding Protein (LBP) [2, 10, 13, 14].The interaction between inducers and sensors leads to the production of mediator compounds derived from plasma proteins or secreted by cells. Mediator compounds can be classified as [2-5, 11, 13-15]: a) Vasoactive amines like histamine and serotonin that act on the vasculature leading to vasodilation or vasoconstriction, affecting also the vascular permeability; b) Vasoactive peptides like substance P, kinins, thrombin or plasmin that also can cause vasodilation and increased vascular permeability; c) Complement Proteins that act by opsonising pathogens surface, i.e. by coating the surface making the pathogens more easily phagocytised. Moreover, they may also create a membrane-attack complex that generate a pore on the pathogen’s lipidic bilayer membrane; d) Proteolytic enzymes that act in order to allow leukocyte migration degrading the extra cellular matrix and the basement-membrane proteins; e) Cytokines that induce local vasodilation, increased blood flow and vascular permeability leading also to leukocytes activation. Cytokines promote as well the influx of cell to the inflammation site; Figure 1: Overview of the inflammatory process and its mediators [15]. 3 f) Chemokines responsible for the chemotaxis that direct the leukocyte traffic towards the inflammation site, acting as directional signals and also by the recruitment of leukocytes; g) Reactive Oxygen Species (ROS) as superoxide, hydroxyl radicals, singlet oxygen, oxygen halides, hydrogen peroxide and Reactive Nitrogen Species (RNS) that interact with lipids, proteins, nucleic acids and other molecules of the pathogens leading to its dead; h) Lipid mediators like eicosanoids and platelet-activating factors. Arachidonic acid (AA) metabolism by cyclooxygenases (COX-1 and COX-2) leads to the formation of eicosanoids, which in turn generate prostaglandins (PGs) that cause vasodilation and are responsible for fever, pain, fatigue, sleepiness, anorexia and social withdrawal, since they act on the central nervous system. The platelet-activating factors, also produced by this pathway, activate several process related to leukocytes activation and vascular regulation. These inflammatory mediators induce the effectors, generally cells, creating the inflammatory response and may also take action as amplifiers of the inflammatory process acting as chemoattractants [3, 14]. Because all the inflammatory components possess numerous elements, different inflammatory pathways can be created so each inflammatory process is unique [5]. The inflammatory process begins within moments from the inducer exposure which explain why, for some, the innate immunity is the most important type of immunity [2, 10, 13]. All these processes are extremely beneficial for the host, however they must be highly regulated since some of them are not able to discriminate between microbial and host cells leading to tissue damages [14]. In order to control the inflammatory process, proinflammatory cytokines are produced such as Tumor Necrosis Factor (TNF) and Interleukin 1 and 6 (IL-1 and IL-6) [2, 4]. Negative feedback mechanisms include the inhibition of proinflammatory signalling cascades, loss of mediator’s receptors, activation of regulatory cells and the production of anti-inflammatory cytokines such as IL-10 and IL-13, Transforming Growth Factor β (TGF-β) and glucocorticoids [2-4, 14]. A balance between pro-inflammatory and anti-inflammatory factors is essential to create an appropriate and effective inflammatory response. When the pathogen or the damaged tissue has been completely removed, the inflammation process should stop allowing the tissue repair and healing. So, when the regulatory process or the acute inflammatory response fails, the process turns into a chronic inflammatory condition. These chronic inflammatory conditions, in turn, lead to host tissue lesions and are implicated in many diseases such as rheumatoid arthritis, atherosclerosis, type 2 diabetes, neurodegenerative diseases asthma, inflammatory bowel diseases, cancer and obesity [2-5]. 1.2 - Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Because of the major role that COX plays both in the acute and chronic inflammatory process, the use of drugs that successfully block this pathway is of extreme importance. Since Non-Steroidal Anti-Inflammatory Drugs have the capability to block COX, even at mM 4 concentrations, they have become an indispensable choice in some chronic inflammatory diseases and in pain management [16, 17]. 1.2.1 Interplay between mechanism of action and therapeutic effects NSAIDs have analgesic, antipyretic, and anti-inflammatory effects being effective drugs in reducing swelling, redness and pain in inflammatory conditions and also general fever and headache. NSAIDs are one of the most effective and prescribed drugs used to treat pain and inflammatory conditions in patients suffering from osteoarthritis, rheumatoid arthritis, gout, low back pain, soft tissue pain and chronic pain [6-9]. NSAIDs act by blocking the cyclooxygenases in a competitive way, inhibiting therefore prostanoids synthesis [7, 12, 18]. COX exists at least in two isoforms: COX-1 and COX-2 [12, 19, 20]. COX-1 and COX-2 are heme-containing bifunctional proteins that convert AA into PG-G2 by a cyclooxygenation reaction and this into PG-H2 by a peroxidase reaction. The activity of tissue-specific synthases and isomerases convert PG-H2 into different prostanoids like PG-E2, PG-D2, PGF2α, prostacyclin (PG-I2) and thromboxane A2 (TX-A2) [12, 19-21]. COX-1 is constitutively expressed being present in almost every tissue and leads to the production of PGs that help in the control of vascular homeostasis, platelet, gastric and renal activity and also protects the gastric mucosa. COX-2 is an inducible cyclooxygenase, active in cells that line blood vessels and that catalysis the production of pro-inflammatory PGs in response to inflammatory factors [6, 12, 19]. Thus, NSAIDs analgesic and anti-inflammatory effects are a consequence of their ability to block COX-2 activity. NSAIDs are characterized in two distinct classes according to their ability to selectively block COX-2 and this is given by the ratio of the IC50 for COX-1 and COX-2 with in vitro studies [22-24]: a) Non-selective COX inhibitors: block both COX-1 and COX-2 in a non-selective manner. They traditionally possess a carboxylic acid (-COOH) as a functional group and represent the most prescribed NSAIDs for fever, pain and inflammation treatment; b) Cox-2 selective inhibitors: selectively block COX-2. Generally they possess a diarylheterocyclic ring as template and less adverse effects. The mechanism of action and the pathways that are blocked by both NSAIDs classes are summarized in Figure 2. Figure 2: NSAIDs mechanism of action and non-selective (traditional) and COX-2 selective inhibited (coxibs) pathways [1]. 5 1.2.2 Interplay between mechanism of action and side effects Despite its beneficial effects NSAIDs, present side effects such as toxicity in the gastrointestinal tract, liver, brain, cardiovascular and renal systems, skin, platelet aggregation and blood pressure [6, 7, 16-18, 25-28]. Bloating, cramping, pain, diarrhoea, constipation, acid reflux, erosins, duodenal and gastric ulcers, perforations, obstructions and bleeding are among the NSAIDs effects on the gastrointestinal tract [6, 8, 16, 18, 29]. The carboxylic acid that generally makes part of the NSAIDs chemical structure leads to gastrointestinal irritation when orally administrated [16, 30]. Besides that, because non-selective COX inhibitors block COX-1 activity, they prevent mucosal cytoprotective PGs formation enhancing gastrointestinal toxicity [8, 9, 19, 20]. NSAIDs effects in renal homeostasis lead to sodium retention, peripheral oedema, weight gain, high blood pressure, congestive heart failure, hyperkaelemia, and acute renal failure. PGs produced for both COX have an important role on renal vasculature regulation and on the electrolyte dynamics, decreasing Na+ reabsorption, so blocking its production leads to deregulation on these processes and ultimately to the effects previously mentioned [6, 16, 19, 31]. COX-2 selective inhibitors toxicity is mostly related to cardiovascular complications like hypertension and increased risk of myocardial infarction and atrial fibrillation. These effects occur due to the absence of important PGs that control platelet-wall interactions, vasoconstriction, platelet aggregation, cholesterol accumulation and that antagonize the effects of hypertensive drugs [6, 7, 16, 19, 23, 24]. 1.2.3 Chemical classification NSAIDs are a very heterogeneous group of molecules with different molecular structures, being the majority organic acids with low water solubility [18, 30, 32-34]. NSAIDs capability to interact with COX-1 and COX-2 is directly related to its chemical properties allowing the carboxylic acid the interaction with COX-1 and having lipophilic drugs a more strong interaction with COX-2 [35]. So, non-selective COX inhibitors generally have carboxylic acids or ketoenolic acids in their constitution while COX-2 selective inhibitors have sulphonamide functional groups on a diarylheterocyclic ring. COX-2 selective inhibitors have been created in order to possess low gastrointestinal adverse effects, reason why their moieties confer them high values of pKa and avoid their interaction with COX-1 [30]. Some NSAIDs properties are summarized on Table1. 6 Table 1: Structure and properties of NSAIDs [32, 36]. Drug Structure pKa Mol wt Log P Non-selective COX inhibitors Aspirin 3.5 180.2 -1.1 Diclofenac 4.2 296.1 4.5 Flurbiprofen 4.22 244.3 4.2 Ibuprofen 4.4 206.3 4.0 Indomethacin 4.2 357.8 4.5 Ketoprofen 4.5 254 ---- Naproxen 4.2 230.3 3.2 Oxaprozin 4.3 293 4.8 COX-2 selective inhibitors Celecoxib 10.7 381.4 3.9 1.2.4 Oxaprozin Oxaprozin (4,5-diphenyl-2-oxazolepropionic acid; Wy-21,743; CAS-21256-18-8) (Table 1) is a non-selective COX inhibitor NSAID with effective anti-inflammatory, analgesic and antipyretic effects. These therapeutic activities derive from its capability to inhibit COX-2 [37-42]. Oxaprozin has an aliphatic propionic acid function attached to the oxazole as side chain, however contrarily to other propionic acid compounds this doesn’t possess a chiral centre [40, 42]. Oxaprozin (Oxa) has a molecular weight of 293 g/mol and presents as a lipophilic white powder, with low water solubility and being slightly soluble in alcohol. It’s a weak acid with pKa around 4.3 with high gastric mucosa tolerance comparatively to other NSAIDs of the same class. That also leads to a slow rate of absorption on the gastric mucosa at stomach pH. Oxaprozin highly binds to plasma proteins (99.9%) with high affinity what leads to a long 7 plasma elimination with a half-life of 50 to 60 hours. This elevated duration of action allows the administration of a single oxaprozin dose per day of 600 to 1800 mg [32, 39, 40, 42, 43]. Oxaprozin like the other NSAIDs and every drug have possible side effects that include stomach pain, constipation, diarrhoea, gas, heartburn, nausea, vomiting and dizziness. More serious side effects include heart attack, stroke, high blood pressure, heart failure (from body swelling due to liquid retention), kidney problems (including kidney failure), stomach and intestine bleeding and ulcers, anaemia, life-threatening skin reactions, life-threatening allergic reactions, liver problems (including liver failure) and asthma attacks (in asthma patients) [44]. 1.3 - NSAIDs delivery systems In order to reduce the adverse side effects of NSAIDs, improve NSAIDs pharmacokinetic properties and to promote a sustained, controlled and targeted drug delivery, several delivery systems have been created in the last years. Next, we will shortly describe some relevant examples of published works in this field. To the date, most of the developed NSAIDs delivery systems are based on polymeric micro/nanospheres. Many NSAIDs have been formulated into microspheres using biodegradable and non-biodegradable polymers and various methods for oral, ocular, parenteral and topical applications Calvo et al. prepared three independent nanocarriers made out of poly-ε-caprolactone loaded with indomethacin, a nanoparticle, a nanocapsule and a nanoemulsion and also a microparticle made out of the same polymer. They verified that these nanosystems had a threefold higher corneal epithelium penetration when compared to a commercial solution, without causing membrane damages. In contrast, the microparticle wasn’t able to increase indomethacin ocular bioavailability [45, 46]. Jayaprakash et al. designed a copolymeric nanoparticle composed by Nisopropylacrylamide, vinyl pyrrolidone and acrylic acid loaded with ketorolac. These nanoparticles were produced in order to increase this NSAID bioavailability. This formulation showed, in vitro, a twofold NSAID permeation and a higher anti-inflammatory activity compared with its aqueous suspension [47]. By a quasi-emulsion solvent technique, Pignatello et al. have been able to produce a polymeric nanoparticle suspension of Eudragit RS 100® loaded with ibuprofen. These nanoparticles allowed a controlled delivery of ibuprofen and an improvement on its ocular availability by increasing its corneal retention and a gradual and prolonged drug release due to its positively charged surface that helps corneal adhesion without ocular tissue toxicity [48]. The same authors have also produced by the same technique a polymeric nanoparticle suspension of Eudragit RS 100® and RL100® loaded with flurbiprofen, in order to also improve its ocular availability. Once again this cationic polymeric nanoparticle suspension indicated a controlled drug delivery and high efficiency when compared to the aqueous drug solution [49]. Eudragit RS100® NSAID loaded nanoparticle were also a matter of study for Adibkia et al.. They loaded these nanoparticles with piroxicam and used them to control inflammatory symptoms in rabbits, concluding that the nanoparticles inhibited the inflammation more successfully than the aqueous suspension of the drug [50]. 8 Polylactic and polyglycolic acid have been used in order to produce nanoparticles loaded with flurbiprofen by Vega et al. and these authors concluded that these nanoparticles were a promising ocular delivery system of drugs due to its excellent ocular tolerance and protection against sodium arachidonate induced inflammation [51]. Badawi et al. loaded indomethacin into chitosan nanoparticles and chitosan nanoemulsion creating positively charged nanocarriers with good corneal affinity and a gradual and longterm (up to 24h) drug release to both external and internal ocular tissues [52]. Contreras-García et al. grafted the co-polymers of N-isopropyl acrylamide (NIPAAm) and N-(3-aminopropyl) methacrylamide hydrochloride (APMA) into a polypropylene film and then loaded the film with NSAIDs in order to analyse their cytocompatibility and frictional properties and as well to evaluate its capability to load and release, for long periods, therapeutic doses of NSAIDs. They concluded that this grafts cold be used in the design of products for biomedical devices in order to promote a controlled NSAIDs release [53]. Alginate microparticles can be used in order to store NSAIDs and deliver NSAIDs, however these microparticles can be destabilised in acidic environments. So in order to create gastroresistent alginate-OCH microparticles, Čalija et al. used an anionic copolymer, Eudragit® L100-55, to stabilize the alginate-OCH microparticles in acidic conditions, leading to a decreased drug release at gastric pH and sustained drug release at intestinal pH. These microparticles may be used for oral NSAIDs delivery due its pH sensitive capacities [54]. Polymeric micelles of poly(ethylene oxide)–poly(β-benzyl L-aspartate) (PEO–PBLA) were used to encapsulate indomethacin by La et al.. The drug released from the micelles was highly pH dependent with rapid release profiles for pH around 7.4 [55]. Giacomelli et al. used poly(2-(dialkylamino)ethyl methacrylate) as micellar core of poly(ethylene oxide)-b-poly[2-(diisopropylamino)-ethyl methacrylate] (PEO-b-PDPA) or poly(glycerolmonomethacrylate)-b-PDPA (PG2MA-b-PDPA) micelles. These micelles allowed a more stable indomethacin encapsulation and prolonged drug retention profiles due to ionic and hydrophobic interactions [56]. Marques et al. formulated nanosized micelles, with different amphiphilic chitosan based polymers in their constitution, loaded with ibuprofen and functionalized with deoxycholic acid (DOCA) and leucine (Leu) moieties. These self-assembled nanoparticles successfully encapsulated the NSAID and delivered it into tumor cells. The anti-tumoral effect of this nanosystem is comparable with that of the traditionally used chemotherapeutics [57]. Roullin et al. prepared biodegradable nanospheres using poly(lactide-co-glycolide) (PLGA). These non toxic nanoparticles showed good NSAIDs encapsulation and local controlled NSAIDs release for up to 48 h. These nanospheres were found to be suitable for oral administration and for prosthesis surface coating [58]. Bédouet et al. created a hydrogel biodegradable microspheres prepared by copolymerization of a methacrylic derivative of ibuprofen with oligo(ethyleneglycol)methacrylate and poly(lactide-co-glycolide-ethylene-glycol) (PLGA-PEG) dimethacrylate as degradable crosslinker. These microspheres were able to prolong the drug release time avoiding the initial burst release, keeping the drug anti-inflammatory activity. So they are suitable for drug delivery by intra-articular injection [59]. Koç et al. designed a class of polyamidoamine (PAMAM) dendrimers for high NSAIDs performance and solubility. For that they introduced a polypropylene oxide as core of the PAMAM (PPO@PAMAM) dendrimers and loaded the dendrimers with ketoprofen, ibuprofen and 9 diflunisal. These formulations augmented NSAIDs solubility, increasing this solubility with the dendrimer concentration and generation in an almost proportional way [60]. Hasegawa et al. reported the creation of a NSAID micellar prodrug using hydrophilic poly(ethylene glycol) (PEG) and a polyacrylamide derivative of ibuprofen as amphiphilic diblock copolymers. Ibuprofen release occurs due to chemical and enzymatic hydrolysis of the micelles and takes place over several days to weeks. These micelles increased ibuprofen solubility and were able to efficiently inhibit two cancer cell lines proliferation [61]. In order to promote corneal residence of indomethacin and consequently augment its ocular availability Balasubramaniam et al. created a Gelrit gellan gum in situ gelling system for topical drop administration. This system was therapeutically effective in rabbits and allowed an in vitro release for up to 8h [62]. Bycopolymerization of poly(hydroxyethyl methacrylate) (pHEMA) with low APMA or 4vinyl-pyridine (VP) proportions Andrade-Vivero et al. created a hydrogel with high NSAIDs loading capacity. Due to this hydrogel properties such as high biocompatibility, thermal stability, acidic and alkaline hydrolysis resistance and easily adaptable mechanical properties, it can be used in oral controlled NSAIDs delivery systems, implants for drug delivery, soft contact lenses (in order to prevent bacterial colonization) or other biomedical devices [63]. In order to provide NSAIDs oral administration Corrente et al. formulated a diclofenac loaded pH sensitive hydrogel using Scl-CM-300 from the scleroglucan polymers class. This hydrogel revealed mucoadhesive properties, affinity to aqueous media, pH sensitive behaviour and biocompatible properties, providing a prolonged drug release time, avoiding the ulcerogenic effects of this NSAID. The authors suggested that this formulation could be used for oral delivery of ulcerogenic NSAIDs or for colon targeting [64]. Saxena et al. created an emulgel (gel emulsion) with Gelucire 39/01 as lipid phase and low viscosity sodium alginate aqueous solution as aqueous phase. These emulgel was loaded with piroxicam for oral administration. These gel jellifies in situ, which prevents the NSAID direct contact with the gastric mucosa avoiding the NSAID side effects while providing sustain drug release [65]. Del Gaudio et al. created almost spherical alginate-based aerogels loaded with ketoprofen with porous texture similar to the related hydrogels. The system responded rapidly to pH changes dissolving the drug. The interesting properties of the aerogel make it suitable for the fast delivery of NSAIDs in the upper gastrointestinal tract which could be useful for NSAID fast delivery for analgesic effects in postoperative pain, dental surgery, renal and uretal acute colic [66]. Sousa et al. produced, by an alumina template assisted synthesis, silica nanotubes with Si-O-H chemical bounds in the inner surface used to create a polycationic surface. The naproxen loaded nanotubes allowed a pH controlled sustainable drug release. The authors suggested the external functionalization of the nanotubes with folate in order to target the pH sensitive delivery systems for the inflamed tissue [67]. Consola et al. described the formation on a cationic vesicle assembled with an amino sugar surfactant and NSAIDs in an aqueous solution. The result was a water soluble cationic vesicle that when used for dermal administration of the NSAIDs showed an improved antiinflammatory activity and prolonged time of NSAIDs residence due to a slower skin diffusion of the system. Besides that, this formulation assembles spontaneously, use biocompatible surfactants and an aqueous vehicle [68]. 16 Nanoparticles drug encapsulation may help to protect the drugs against the harsh GI environment (pH and enzymes) and also increase mucoadhesion and retention in the GI tract leading to an improved drug uptake [109]. 1.7 Specific aims and strategy In this context the main objective of this work, is to develop an innovative oxaprozin delivery system, based on lipid nanoparticles, with the main purpose of overcoming the most deleterious side effects of this drug – gastric disorders. Thereby, the main steps included during the first semester, literary research, state of the art and work plan elaboration. The second part, the research work, was fulfilled during the second semester at the Department of Chemical Sciences at the Faculty of Pharmacy, University of Porto. Research work consisted first on the production of oxaprozin loaded lipid that embodies the selection of an appropriate lipid mixture according to the solubility of the drug, the optimization of the production method and of the lipid phase constitution according to the encapsulation efficiency, loading capacity, particles diameter and zeta potential. The second step was surface functionalization with Folic Acid (FA), this step involved the synthesis of a DSPE-PEG2000-FA conjugate using DSPE-PEG2000-NH2 and folic acid. Third step was the characterization of the developed drug delivery systems by experiments of particle size and zeta potential using dynamic light scattering (DLS) and electrophoretic light scattering (ELS) respectively, encapsulation efficiency, loading capacity and functionalization analysis using UV-Vis spectroscopy and in vitro release studies at physiological conditions. The final step was the validation of the developed drug delivery systems by cell viability and cytotoxicity studies in Caco-2 cell line using Methylthiazolyldiphenyl-tetratozium Bromide (MTT) and Lactase Dyhydrogenase (LDH) and permeability studies in Caco-2 cell lines by UV-Vis spectroscopy quantification. This approach could improve the NSAIDs use by reducing its serious side-effects, promoting its controlled release and drug action in inflamed regions. 17 Chapter 2 Materials and Methods 2.1 - Materials The lipids Apifil, Cetyl Palmitate, Compritol HD5 A, Gelucire 33/01 Lipocire CM, Precirol Ato 5 and Superpolystrate were a kind gift from Gattefossé (Gattefossé, France) and the lipids Imwitor 308, Softisan 100, Witepsol E76, Witepsol E85, Witepsol H32, Witepsol S51 and Witepsol S58 were gently offered by Sasol (Sasol, Germany). Miglyol 812 was purchased from Acofarma® (Terrassa, Spain). Tween® 60, TritonTM X-100, Thiazolyl Blue Tetrazolium (MTT), Trypan Blue powder, dimethyl sulfoxide ≥99.9%, oxaprozin (4,5-diphenyl-2-oxazolepropanoic acid; Daypro), acetic acid ≥99.8%, potassium phosphate monobasic, folic acid, triethylamine, dicyclohexylcarbodiimide, N-hydroxysuccinimide and Dulbecco’s Phosphate Buffered Saline pH 7.4 (PBS) were obtained from Sigma-Aldrich®. Oleic acid was from May & Baker Ltd (England). Sodium phosphate monobasic monohydrated was acquired from Fluka (Germany). Sodium chloride was purchased from Panreac (Spain). Sodium acetate was from Fluka Analytical (Japan). Sodium hydroxide was obtained from VWR International (Belgium). Acetonitrile (Lichrosolv®) were obtained from MERK (Germany). DSPE-PEG.NH2 (1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt)) was purchased from (Avanti® Polar Lipids Inc.; Alabaster, Alabama, USA). SIF Powder was obtained from Biorelevant.com (Croydon, Surrey, UK). LDH Cytotoxicity Detection Kit was from Takara Bio Inc. (Shiga, Japan). Hanks’ Balanced Salt Solution [-] CaCl2, [-] MgCl2 (HBSS), Dulbecco’s Modified Eagle’s Medium (DMEM) + GlutaMAXTM-I, 0.25% Trypsin-EDTA (1X), Penicillin-Streptomycin (Pen Strep) and Heat Inactivated Fetal Bovine Serum (FBS) (origin: South America) were purchased from Gibco® by Life TechnologiesTM (UK). All the weighting measurements performed using a Kern ACS-80-4 digital analytical balance (Kern & Sohn; Balingen, Germany). pH measurement were obtained using a Crison pH meter GLP22 with a Crison 52-02 tip (Crison; Barcelona, Spain). Ultrapure water was purified by an Ultra-pure water system (Milli-Q, Sartorius, Arium® pro, Sartorius Weighing Technology; Gettingen, Germany, Filters: Sartorius Arium® Cartige 1 and 2, Sartorius Stedin Biotech; Gettingen, Germany) by a reverse osmosis process. 2.2 - Methods 2.2.1 Choice of solid lipid for improved drug loading Drug incorporation on a nanosystem is influenced by its capacity to dissolve on the system components [98, 103]. Thus, LNs drug solubility plays a major role, making the choice of lipids an importance step, in order to have a lipid or lipids mixture where the drug exhibit high solubility. 18 The analysis of oxaprozin qualitative solubility in the lipids was performed by melting an equal amount of different commercial lipids: Apifil, Cetyl Palmitate, Compritol HD5 A, Gelucire 33/01, Imwitor 308, Lipocire CM, Precirol Ato 5, Softisan 100, Superpolystrate, Witepsol E76, Witepsol E85, Witepsol H32, Witepsol S51 and Witepsol S58. The melting process was realized by slowly augmenting the temperature from 50ºC to 70ºC in order to assess the approximated phase transition temperature of the lipids. After the melting process, an amount of oxaprozin (always the same) was added to the melted lipids in order to qualitatively infer oxaprozin’s solubility on the different lipids. 2.2.2 Lipid nanoparticles synthesis Lipid nanoparticles were prepared by a modified free organic-solvent emulsification/sonication method combining high shear homogenization in an ultra-turrax, where a “pre-emulsion” with particles in the micrometer range are produced, followed by an ultra-sonication that will reduce the microparticles into nanoparticles resulting in a nanoemulsion. Cooling of the nanoemulsion allows lipid crystallization and the formation of the lipid nanosuspension [119]. Briefly, the lipid phase containing the desired lipid or lipids combination and the stabilizer Tween 60 was melted at a temperature above the phase transition temperature with or without stirring for better oxaprozin dissolution. Heating without stirring was performed in a water-bath (Medingen E5 Bath-Thermostat) and heating under stirring was performed in a top stirring hotplate (Barnstead International Thermolyne Cimarec® 2; Iowa, USA). For the synthesis of oxaprozin loaded lipid nanoparticles, oxaprozin was added to the lipid phase before the melting process. The molten lipids are dispersed in a specific amount of an aqueous phase at the same temperature (ultrapure water) by high-speed stirring in an ultra-turrax (Ystral X10/20 E3; Ballrechten-Dottingen, Germany) followed by sonication (Sonics and Material Vibra-CellTM with a CV-18 probe; Newtown CT, USA). The solutions are cooled at room temperature and in some cases an extra amount of the aqueous phase at room temperature is added. The formulations are then stored at room temperature. During the optimization process several lipids conjugations, aqueous phase quantities, stirring times during heat, high speed stirring and sonication parameters (time and potency) and post-synthesis aqueous phase addition quantities were tested in order to establish the best conditions for the lipid nanoparticles production. The optimized formulation is synthesised by melting, under 10 minutes stirring, the lipid phase constituted by the solid lipid Precirol Ato 5 (170 mg), the liquid lipid Miglyol 182 (90 mg) and the surfactant Tween 60 (60 mg). For the production of oxaprozin loaded LNs, 30 mg of oxaprozin are added to the lipid phase and this amount is deducted from the total amount of solid lipid. 4.4mL of heated ultrapure water are added to the lipid phase and the emulsion mixed by ultra-turrax (30 seconds at 7000 RPM) followed by ultrasonication (5 minutes at 70% intensity). 4.4 mL of room temperature ultrapure water is then added and the emulsion cooled at room temperature. 19 2.2.3 Synthesis of disteroylphosphatidylethanolamine-poly(ethylene glycol)-folic acid (DSPE-PEG-FA) conjugate DSPE-PEG2000-FA conjugate synthesis can be summarized in four main steps: activation of the folic acid, coupling to DSPE-PEG2000-NH2, purification and lyophilisation. N-Hydroxysuccinimide ester of folic acid (NHS-FA) was prepared by dissolving, under anhydrous conditions through overnight stirring in the dark, 1.0 g of folic acid (FA) into a mixture of 40 mL of anhydrous dimethyl sulfoxide (DMSO) and 0.5 mL of triethylamine (TEA). FA was then mixed with 0.5 g of dicyclohexylcarbodiimide (DCC) and 0.52 g of Nhydroxysuccinimide (NHS) and stirred for further 18 hours in the dark. The resulting solution was then filtered with a 0.45 μM filter in order to remove the precipitated side product (dicylcohexylurea (DCU)), DMSO and TEA were evaporated under vacuum [120, 121]. Coupling of the FA-NHS stock solution with DSPE-PEG2000-NH2 was accomplished by overnight stirring in the dark under anhydrous conditions of 2 mL of the resulting solution of activated folate with 50 mg of DSPE-PEG2000-NH2 dissolved in 1 ml of DMSO. After the process DMSO of removed by evaporation under vacuum and addition of 6 mL of water [121]. DSPE-PEG2000-FA conjugate was then dialyzed against 500 mL of ultrapure water using a dialysis membrane (Cellu.Sep®T1, 3500 NMWCO, Membrane Filtration Products, Inc.; Seguin, TX, USA) for 48 hours in order to remove the unconjugated FA [120]. The resulting solution was then lyophilized (Virtis Sentry 2.0 lyophilizator, SP Scientific) forming a yellow dry powder, the final DSPE-PEG2000-FA conjugate, stored at 4ºC. 2.2.4 Synthesis of DSPE-PEG-FA functionalized LNs Functionalization of the synthesised LNs with DSPE-PEG-FA was obtained by adding the DSPE-PEG2000-FA on the lipid phase in order for it to be encapsulated in the LNs matrix. The encapsulated DSPE-PEG2000-FA will be randomly placed among the nanoparticle so that despite a part of the encapsulated DSPE-PEG2000-FA may be placed on the interior of the particle a part of this DSPE-PEG2000-FA will also be placed on the surface of the particle allowing an active macrophages targeting. DSPE-PEG2000-FA used to produce functionalized LNs was optimized and for the production of the final formulation, functionalized nanoparticles were obtained by adding 2% of the total lipid mass (lipids and surfactant) of DSPE-PEG2000-FA to the lipid phase. 2.2.5 LNs characterization 2.2.5.1 Particle size measurements Particles in suspension present movement (Brownian movement), Dynamic Light Scattering (DLS), also known as photon correlation spectroscopy, use this movement in order to obtain the mean particles diameter and diameters distribution allowing also the identification of the different size populations present in solution. Focusing a laser on the suspension, the movement of the particles will lead to variations on the light diffraction over time. These variations vary with the diffusion coefficient of the medium and with the particles size. Since bigger particles have slower movements than smaller particles they will diffract more the light. So analysing the movement of the particles by the light deviation and knowing the temperature and viscosity of the sample, DLS can calculate the hydrodynamic diameter of the particles on the solution [122]. 20 The produced nanoparticles were characterized by its mean hydrodynamic diameter and size distribution (polydispersity index) in a Particle Size Analyzer (Brookhaven Instruments Corporation; Software: Particle Sizing v.5 Brookhaven Instruments; Holtsville, NY, USA) operating at a scattering angle of 90º, at 37ºC, with dust cut-off set to 30 and refractive index of the particles set to 1.33. Measurements were performed by 6 runs of 2 minutes each and the mean size and polydispersity of the formulations were obtained. Before the measurements the formulations were diluted (1:800) and filtered with a single use syringe filter (Ministart, pore size 800 nm, Satorius Stedin Biotech; Goettingen, Germany). 2.2.5.2 Zeta Potential measurements Zeta potential is a physical property exhibited by particles in suspension that determines its stability. Particles surface charge promotes interactions with the surrounding ions creating an unequal distribution of the solution ions in close proximity to the particle. This distribution leads to the formation of an inner zone were the solution ions are powerfully bonded to the surface and a more outer zone with a weakest connection. On this outer region the particle and the ions form a stable entity and the zeta potential is the potential associated at this theoretical limit [123]. Since particles with the same charge tend to repel themselves and since this potential is strictly connected to the particle surface charge, zeta potential is correlated with the suspension stability having particles with higher zeta potential, in modulus, higher tendency to repel themselves and consequently higher stability [124]. One can generally assume that particles have great stability for zeta potential values with absolute value superior to 30 mV [123, 124]. Electrophoretic Light Scattering (ELS) is a method that calculates the zeta potential by light scattering. Two electrodes are inserted on the formulation creating an electric potential that attracts to the electrodes particles of the opposite charge. The movement created by this migration is analysed and the particles velocity measured by the light deviation, with this parameter is possible to calculate the particles zeta potential [125, 126]. Characterization of the particles zeta potential was performed in a Zeta Potential Analyser (ZetaPALS, Brookhaven Instruments Corporation, Software: PALS Zeta Potential Analyser v.5, Brookhaven Instruments; Holtsville, NY, USA) operating at a scattering angle of 90º, at 37ºC. Measurements were performed by 6 runs of 10 cycles each and the zeta potential and standard deviation of the formulations were obtained. Before the measurements the formulations were diluted (1:800) and filtered with a syringe filter (800 nm). 2.2.5.3 Oxaprozin Encapsulation Efficiency (EE) Oxaprozin encapsulation efficiency was determined by calculating the difference between the total amount of used oxaprozin to produce the formulations and the amount of free oxaprozin remaining on the aqueous phase. Formulations where diluted in ultrapure water (1:50) and centrifuged in Amicom® Ultra Centrifugal Filter Devices, ultracel®-50k (50000 NMWL) (MERK Milipore, Ltd; Cork, Ireland). Centrifugation (Heraeus Multifuge X1R centrifuge, Thermo Fisher Scientific; Germany) was performed with spin at 2664g for 40 minutes at 20ºC. Free oxaprozin was present in the supernatant and was quantified by UV-Vis spectroscopy using a V-660 spectrophotometer 21 (Jasco Corporation, Software: Spectra Manager v.2, Jasco Corporation; Easton, MD, USA) at 285 nm. Encapsulation efficiency was calculated as follows (Equation 1): Equation 1: encapsulation Efficiency calculation. 2.2.5.4 Loading capacity (LC) LNs loading capacity was calculated using the oxaprozin encapsulation efficiency as follows (Equation 2): Equation 2: Loading capacity calculation. 2.2.5.5 Transmission electron microscopy (TEM) In TEM thin samples are illuminated by an accelerated electrons beam and the electrons that are able to pass through the sample go into a system of electromagnetic lenses that enlarge and focus the image that is then recorded. Samples were prepared by diluting the formulations in ultrapure water (1:50) and then 10 μL of this solution placed on a cooper-mesh grid and let to rest for 2 minutes at room temperature after which the water excess was removed. For contrasting, 10 μL of 0.75% uranyl acetate solution was placed on the grid surface and let to rest for 30 seconds at room temperature. Solution excess was removed and the samples observed in a JEM-1400 Transmission Electron Microscope (JEOL Ltd., USA) with an accelerating voltage of 80 kV. 2.2.6 In vitro release studies In vitro release studies were performed using a cellulose dialysis bag diffusion technique (Cellu.Sep®T1, 3500 NMWCO, Membrane Filtration Products, Inc.; Seguin, TX, USA) filled with 2 mL of the formulation (functionalized or non-functionalized oxaprozine loaded nanoparticles, 1:8 in ultrapure water). In order to mimic the particles path in the body after oral administration, samples were incubated at 37ºC under gentile stirring (IKA®-Werke RT15-P Hot Stirring Plate; Germany). To simulate the transit from stomach to intestine samples were incubated first for 3 hours in 80 mL of fasted state simulated gastric fluid (FaSSGF: NaCl/HCl solution, pH 1.2 with SIF® Powder) and then placed for 4 hours in 80 mL of fasted state simulated intestinal fluid (FaSSIF: buffer solution containing potassium dihydrogen phosphate, pH 6.5 with SIF® Powder). From this point some samples were placed in 80 ml of simulated physiologic fluid (buffer solution containing monopotassium phosphate, pH 7.4) until the end of the experience in order to simulate the particles that never reach the inflamed regions. Other samples were placed until the end of the experience in simulated inflammatory fluid (buffer solution containing acetic acid and sodium acetate, pH 5.4) to simulate the release profile of the LNs in inflamed regions. Finally the rest of the samples were placed for 18 hours in the simulated physiologic fluid and then in the simulated inflammatory fluid until the end of the 22 experience in order to mimic the release profile of LNs that circulate on the blood for a considerable period of time until reaching the inflamed regions. At regular intervals, aliquots were collected for a UV-Vis microplate (UV flat bottom Microliter® plates, Thermo Electron Corporation; Vantaa, Finland) and replaced by an equal amount of fresh fluid (correspondent to the one that was being used). Oxaprozin release was analysed by UV-Vis spectroscopy using a microplate reader (BioTek Instruments Inc., Synergy HT, Software: Gen5 v1.08.4, BioTek Instruments Inc.; Winooski, USA) at 285 nm. 2.2.7 Caco-2 cell culture Caco-2 cells (P.23 to P.32) were cultured at 37ºC in an 5% CO2 atmosphere (Unitherm CO2 Incubator 3503 Uniequip; Planegg, Germany) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (Pen Strep). For every two to three days cells were washed with HBSS and the medium changed. Cells were subcultured at 80 to 90% confluence by chemical detachment with trypsin-EDTA and cells counted in a Neubauer chamber (Improved Neubauer Bright-line, Boeco; Germany) with 25% (v/v) Trypan Blue solution (0.4% (w/v) in PBS) to exclude non-viable cells. Cells were then seeded at a density of 750000 cells per 75 cm2 flasks (Tissue culture flasks 250 mL (75 cm2), 0.2 μm vented plug seal cap, Falcon®, Becton Dickson; England) in 10 mL of DMEM. 2.2.8 Cell viability assay The effect of the designed LNs on cell viability was measured using the methylthiazolyldiphenyl-tetratozium bromide (MTT) conversion assay. Caco-2 cells were seeded in 96-well tissue culture test microplates (Tissue Culture OrPlates, surface treated flat bottom, Orange Scientific; Belgium) at a density of 10000 cells/well in 100 μL supplemented DMEM medium and incubated for 22 hours at 37ºC in an 5% CO2 atmosphere. 100 μL of different concentrations (5 μM, 10 μM, 50 μM, 100 μM, 500 μM and 1000 μM) of free oxaprozin, oxaprozin loaded functionalized and non-functionalized LNs and the correspondent amounts of placebo functionalized and non-functionalized LNs, as well as the positive control (culture medium) and the negative control (Triton X-100 1% (v/v) in PBS) were then added and let to incubate with the cells for 4 hours. Supernatant was transferred for a 96-well tissue culture test microplate and stored for the realization of the cell toxicity study. 200 μL of a MTT solution (5 mg/mL MTT in PBS stock solution diluted to a final concentration of 0.5 mg/mL in culture medium) were then added to each cell seeded well and incubated for 3 hours at 37ºC in an 5% CO2 atmosphere to allow the formation of formazan crystals. MTT was then rejected by plate inversion and 200 μL of DMSO were added and incubated for 15 minutes at 37ºC in a 5% CO2 atmosphere protected from light in order to elute blue formazan from cells and solubilise the formazan crystals. Absorbance was read using a microplate reader at 550 nm and 690 nm for background subtraction. Cell viability was determined according as follows (Equation 3): Equation 3: Cell viability calculation. 23 2.2.9 Cell toxicity assay The cell toxicity assay realized analyzes the presence of Lactase Dyhydrogenase (LDH) on the culture medium. LDH is a cytoplasmatic enzyme present in most cells and is released to the culture medium in cells with cytoplasmatic membrane damages. Using a LDH Cytotoxicity Detection Kit, which will react with the LDH forming formazan, one can quantify the formazan which can be directly correlated with the LDH present on the culture medium assessing the cell death. The first steps of the LDH assay are equal to those performed on the cell viability assay until the end of the incubation period of the samples with the cells. From here on the LDH assay uses the recovered supernatant that resulted from the incubation. The recovered microplates were centrifuged (Sigma 3k-2 microplates centrifuge) in order to deposit the cellular remains for 10 minutes at 250g, at room temperature. After centrifugation, 100 μL of the supernatant were carefully transferred for another 96-well tissue culture test microplate and 100 μL of the LDH Cytotoxicity Detection Kit reaction mixture added to the wells and let to incubate in the dark for 15 minutes at room temperature. Absorbance was then read using a microplate reader at 490 nm and 690 for background subtraction and cytotoxicity calculated having into consideration that for this assay the positive control were the cells treated with 1% Triton X-100 (v/v) in PBS and the negative control were the cells treated with culture medium. Cell cytotoxicity was determined according as follows (Equation 4): Equation 4: Cell cytotoxicity calculation. 2.2.10 Cell permeability studies Caco-2 cells were seeded on Transwell diffusion cells with a density of 100000 cells per cm2 (45000 cells per insert of 4.67 cm2 polycarbonate membrane with 3.0 μm porosity) in supplemented DMEM culture medium and maintained in culture medium for 21 days. After 21 days of culture, Caco-2 cells form an absorptive polarized monolayer developing an apical brush border and enzyme segregation, so that they can be used to mimic the intestinal endothelium [127]. For every three to four days cells were observed on an trinocular inverted microscope (Motic AE2000 TRI coupled with a camera Moticam 5 MP; Spain) and the transepithelial electrical resistance (TEER) measured on the polycarbonate filters of the Transwell diffusion cells using an epithelial voltammeter (Epithelial voltohmmeter EVOM2, World Precision Instruments, Inc.; USA) to evaluate thigh junctions formation. Cells were also washed twice with PBS and the culture medium substituted. At the 21st day TEER must be over 200 Ω.cm2, the reference value which indicates that the monolayer presents characteristics similar to those of the intestinal mucosa [128]. For the permeability studies the culture medium was removed and the cells, the insert and receptor well washed twice with PBS. Fasted state simulated intestinal fluid (FaSSIF: buffer solution containing potassium dihydrogen phosphate, pH 6.5 with SIF® Powder) and the reference medium PBS were added to the correspondent inserts and receptor wells. Free oxaprozin and functionalized and non-functionalized oxaprozin loaded LNs at 100 μM concentration (determined by the cell viability and cytotoxicity assays) diluted on the correspondent medium were incubated on the inserts for 4 hours at 37ºC in a 5% CO2 atmosphere. At different times, 160 μL aliquots were collected for a 96-well UV-Vis 24 microplate from the receptor well and substituted by the same amount of the correspondent medium, along with aliquots from the insert at the initial and final time. TEER measurements were also performed at the same times in order to evaluate cell viability and the opening of the tight junctions during the 4 hours assay. At the end of the study, 40 μL of a mixture of acetonitrile (92%) and acetic acid (8%) were added at the aliquots in order to dissolve the lipid matrix of the LNs and expose oxaprozin in the solution to be quantified. Oxaprozin quantification was performed by UV-Vis spectroscopy using a microplate reader, at wavelength of 285 nm. Apparent permeability coefficient (Papp) was calculated as follows (Equation 5): Equation 5: Apparent permeability coefficient calculation. Where Q is the total amount of permeated oxaprozin (μg), A is the diffusion area (cm2), C0 is the initial oxaprozin concentration (μg/mL) and t is the time of the experience (s). 2.2.11 Statistical analysis Statistical analyses were performed using IBM® SPSS® Statistics software (v.22.0.0.0; IBM, Armonk, NY, USA). The measurements were repeated at least three times and data expressed as mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) and differences between groups compared by Bonferroni and Tukey post-hoc tests with a P value of <0.05 considered statistically significant. 25 Chapter 3 Results and Discussion 3.1 - Choice of solid lipid for improved drug loading Drug solubility in the lipid phase of the lipid nanoparticles plays an important role on the entrapment efficiency, drug loading and on the release profile of the lipid nanoparticles. Phase transition temperature is also an important feature since it correlated with the stability of the crystalline structure of the lipid nanoparticle. Table 2 presents the lipids that were tested, their phase transition temperature and also their relative ability to solubilise oxaprozin. Table 2: Lipids phase transition temperature and qualitative oxaprozin dissolution capacity. Lipid Liquid at Oxaprozin dissolution capacity 50° C 60° C 70° C Apifil    - Cetyl Palmitate    - Compritol HD5 A    + Gelucire 33/01    - Imwitor 308    +++ Lipocire CM    ++ Precirol Ato 5    + Softisan 100    - Superpolystrate    +++ Witepsol E76    - Witepsol E85    + Witepsol H32    - Witepsol S51    - Witepsol S58    ++ Imwitor 308 and Superpolystrate with phase transition temperature inferior to 50ºC were the only lipids with a very good apparent oxaprozin dissolution capacity followed by Lipocire CM and Witepsol S58 with phase transition temperature around 50ºC with good oxaprozin solubilisation and Compritol HD5 A, Precirol Ato 5 with phase transition temperature around 70ºC and Witepsol E85 with phase transition temperature close to 50ºC shown low oxaprozin solubility. The other lipids were not able to solubilise oxaprozin. 32 LN36 NLC Precirol Ato 5 350 30 4.4 12000 120 70 15 -- 802 ± 54 0.40 ± 0.01 -32.2 ± 0.5 -- -- Miglyol 182 150 Tween 60 100 LN37 NLC Precirol Ato 5 330 20 4.4 12000 120 70 15 4.4 531 ± 21 0.320 ± 0.007 -29.4 ± 0.8 96.1 3.3 Miglyol 182 150 Tween 60 100 Oxaprozin 20 LN38 NLC Precirol Ato 5 180 10 8.8 7000 30 70 5 -- 340 ± 8 0.219 ± 0.004 -33.5 ± 0.9 -- -- Miglyol 182 90 Tween 60 60 Oxaprozin 20 LN39 NLC Precirol Ato 5 180 10 8.8 7000 30 70 5 -- 425 ± 10 0.318 ± 0.006 -32.3 ± 0.5 -- -- Oleic Acid 90 Tween 60 60 Oxaprozin 20 LN40 NLC Precirol Ato 5 200 10 4.4 7000 30 70 5 4.4 375 ± 5 0.19 ± 0.01 -- -- -- Miglyol 182 90 Tween 60 60 LN41 SLN Cetyl Palmitate 270 10 8.8 7000 30 70 5 -- 241 ± 7 0.121 ± 0.005 -61 ± 3 97.8 5.9 Tween 60 60 Oxaprozin 20 LN42 SLN Cetyl Palmitate 290 10 8.8 7000 30 70 5 -- 264 ± 1 0.16 ± 0.02 -52.6 ± 0.6 -- -- Tween 60 60 LN43 NLC Precirol Ato 5 180 10 4.4 7000 30 70 5 4.4 217 ± 5 0.120 ± 0.004 -44.7 ± 0.9 95.8 5.8 Miglyol 182 90 Tween 60 60 Oxaprozin 20 33 LN44 NLC Precirol Ato 5 200 10 4.4 7000 30 70 5 4.4 211.9 ± 4 0.11 ± 0.01 -55.6 ± 0.6 -- -- Miglyol 182 90 Tween 60 60 LN45 SLN Cetyl Palmitate 260 10 8.8 7000 30 70 5 -- 244 ± 4 0.07 ± 0.01 -32.1 ± 0.9 98.9 9.3 Tween 60 60 Oxaprozin 30 LN46 NLC Precirol Ato 5 170 10 4.4 7000 30 70 5 4.4 245 ± 4 0.14 ± 0.01 -28.1 ± 0.9 97.5 9.1 Miglyol 182 90 Tween 60 60 Oxaprozin 30 LN47 SLN Cetyl Palmitate 266.7 10 8.8 7000 30 70 5 -- 250 ± 3 0.06 ± 0.02 -31.8 ± 0.4 95.2 5.8 Tween 60 60 Oxaprozin 20 DSPE-PEG2000-FA 3.3 LN48 NLC Precirol Ato 5 176.7 10 4.4 7000 30 70 5 4.4 211 ± 3 0.174 ± 0.007 -33 ± 1 83.6 5.1 Miglyol 182 90 Tween 60 60 Oxaprozin 20 DSPE-PEG2000-FA 3.3 LN49 SLN Cetyl Palmitate 263.4 10 8.8 7000 30 70 5 -- 248 ± 5 0.06 ± 0.02 -27 ± 1 94.9 5.7 Tween 60 60 Oxaprozin 20 DSPE-PEG2000-FA 6.6 34 LN50 NLC Precirol Ato 5 193.4 10 4.4 7000 30 70 5 4.4 204 ± 3 0.17 ± 0.02 -32.5 ± 0.7 92.4 5.3 Miglyol 182 90 Tween 60 60 Oxaprozin 20 DSPE-PEG2000-FA 6.6 LN51 NLC Precirol Ato 5 168 10 4.4 7000 30 70 5 4.4 237 ± 4 0.185 ± 0.007 -29.3 ± 0.7 88.3 6.8 Miglyol 182 90 Tween 60 60 Oxaprozin 25 DSPE-PEG2000-FA 7 LN52 NLC Precirol Ato 5 163 10 4.4 7000 30 70 5 4.4 221 ± 4 0.169 ± 0.005 -35 ± 1 93.1 8.7 Miglyol 182 90 Tween 60 60 Oxaprozin 30 DSPE-PEG2000-FA 7 LN53 NLC Precirol Ato 5 170 10 4.4 7000 30 70 5 4.4 251 ± 11 0.19 ± 0.01 -22.6 ± 0.9 84.1 7.6 Miglyol 182 90 Tween 60 60 Oxaprozin 30 DSPE-PEG2000-FA 14 LN54 NLC Precirol Ato 5 170 10 4.4 7000 30 70 5 4.4 280 ± 14 0.16 ± 0.01 -23.1 ± 0.5 86.1 7.8 Miglyol 182 90 Tween 60 60 Oxaprozin 30 DSPE-PEG2000-FA 10.5 35 LN55 NLC Precirol Ato 5 170 10 4.4 7000 30 70 5 4.4 253 ± 6 0.255 ± 0.007 -31.3 ± 0.6 91.2 8.4 Miglyol 182 90 Tween 60 60 Oxaprozin 30 DSPE-PEG2000-FA 7 36 3.3 - Nanoparticles characterization In order to ensure that the developed formulations possessed the desired and necessary characteristics to allow a controlled and targeted oxaprozin release the lipid nanoparticle that resulted from the optimization process was then characterized along with its correspondent’s placebo and non-functionalized particles. The first indicator of a successful functionalization process and of the synthesis of a workable liquid suspension was visual confirmation. From Figure 4 we can verify the formation of a milky suspension characteristic of the ultra-sonication lipid nanoparticle synthesis method. These suspensions were of low viscosity which is essential to its use, having the formulations with oxaprozin a slightly higher viscosity. It is also possible to notice colour change in the functionalized nanoparticles when compared with the non-functionalized correspondents. This colour alteration is the first indicative of a successful functionalization of the formulations. Oxaprozin deposits were not visible however small DSPE-PEG2000-FA deposits were visible tending to disappear over time and with agitation which may indicate suspension stabilization over time. a) b) c) d) Figure 4: Lipid nanoparticles synthesised by emulsification-sonication: a) Non-functionalized NLCs placebo, b) Non-functionalized NLCs + Oxa, c) Functionalized NLCS Placebo, d) Functionalized NLCs + Oxa. 37 Nanoparticles diameter, polydispersity, zeta potential, encaplsulation efficiency and loading capacity were also evaluated and it is summarized on the Table 4. Table 4: Physicochemical properties of the developed LNs. Mean effective diameter, polydispersity, zeta potential, encapsulation efficiency and loading capacity of placebo and oxaprozin non-functionalized and folate functionalized LNs. Values represent mean ± SD (n=3); a) P<0.05 relatively to the correspondent NLCs placebo formulation; b) P<0.05 relatively to the non-functionalized NLCs formulation. Non-funcionalized NLCs Placebo Non-funcionalized NLCs + Oxa Funcionalized NLCs Placebo Funcionalized NLCs + Oxa Diameter (nm) 216 ± 23 289 ± 27a 234 ± 24 281± 25a Polydispersity 0.28 ± 0.04 0.17 ± 0.03 0.23 ± 0.05b 0.14 ± 0.03 Zeta Potential (mV) -41 ± 2 -44 ± 2 -41 ± 2 -42 ± 2 Encapsulation Efficiency (%) - 97.6 ± 0.3 - 95.1 ± 0.5b Loading Capacity (%) - 9.1 ± 0.3 - 8.7 ± 0.5b Considering the size and polydispersity of our nanoparticles, it is necessary to have into consideration that they were obtained using filtered nanoparticles. The filtration process was necessary in order to reduce the deviating effect that bigger particles cause in the diameter reading process, this also allowed to ensure that even the smallest particles had the desired diameter. From the obtained data Table 4 one can conclude that the NLCs diameters were all greater than 200 nm, meaning that our nanoparticles can be recognized be the macrophages as desired [77, 79]. Despite the fact that the functionalization process did not interfered with the diameter of the nanoparticles, the oxaprozin encapsulation leads to the formation of nanoparticles of large diameter than the corresponding placebo which can be a prove that the oxaprozin is in fact being encapsulated and may help to stabilize the lipids aggregates during the synthesis. The polydispersity of the formulations were around 0.2, meaning that formulations are constituted by a well-defined and almost monodisperse population of nanoparticles with low variability typical of LNs produced using the high shear homogenization and ultrasound method [129]. These values also suggest a uniform functionalization process that leads to a small decrease on the polydispersity. Zeta potential of the particles that constitute the formulations is greater in modulus to 40 mV ensuring the stability of our formulations and revealing that they do not tend to form aggregates since they are superior to the reference value of |30| mV [123, 124]. Oxaprozin encapsulation and the functionalization process did not interfered with the zeta potential values and consequently with the stability of the formulations. The encapsulation efficiency and consequently the loading capacity are influenced by the functionalization process, leading to slightly lower values of these parameters. However the obtained values are good and very satisfactory revealing the high encapsulation efficiency and loading capacity of these nanoparticles. Transmission electron microscopy (TEM) was also used to analyse the formulations. Transmission electron micrographs (Figure 5) revealed the spherical shape of the nanoparticles and that the encapsulation and functionalization process did not to considerably affect the particles shape. The micrographs shown also particles with sizes slightly different from those obtained by DLS. Though, most of the particles, primarily from the functionalized oxaprozin loaded NLC (the formulation of interest) demonstrated sizes 38 superior to 200 nm which is the minimum size pretended for our particles. Diameter differences between TEM and DLS analysis can be justified by the fact that DLS analyses the hydrodynamic radius. Moreover DLS provides a mean size of the populations present in the formulation while TEM provides a direct size of the particles. UV-Vis spectophotometric analysis of the functionalized LNs after being destroyed revealed a peak on the Placebo LN similar to those found on free functionalization agent, DSPE-PEG2000-FA, (Figure 6) on the other hand in oxaprozin loaded LNs due to the presence of the oxaprozin with a broad peak near the functionalization agent peak one cannot clearly identify the functionalization agent peak (Figure 7). However the qualitative analysis can prove the presence of the functionalization agent on the LNs. a) b) c) d) Figure 5: TEM micographs: a) Non-functionalized NLCs placebo, Magnification 40000X. b) Non-functionalized NLCs + Oxa, Magnification 40000X. c) Functionalized NLCs Placebo, Magnification 40000X. d) Functionalized NLCs + Oxa, Magnification 12000X. 39 Figure 6: UV-Vis analysis of the functionalization process in Functionalized NLCs placebo. Figure 7: UV-Vis analysis of the functionalization process on Functionalized NLCs + oxaprozin. 3.4 - In vitro release studies The in vitro oxaprozin release study was designed in order to simulate the lipid nanoparticles path through the body after oral administration. Particles were placed at body temperature (37ºC) in gastric medium for 3 hours and then 4 hours in intestinal medium in order to simulate the gastrointestinal transit. Then the particles were placed some in physiologic medium in order to simulate particles that never reach the inflamed region and stood in circulation, some in inflammatory medium to simulate particles that immediately accumulate in inflamed regions and some 18h hours in physiologic medium and then in 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 200 250 300 350 400 450 Abs nm DSPE-PEG2000-FA Functionalized NLCs Placebo 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 200 250 300 350 400 450 Abs nm Free oxaprozin Functionalized NLCs + Oxa 40 inflammatory medium to reproduce the path of particles trough the blood and its later placement in inflamed regions. Figure 9: Oxaprozin release in gastric, intestinal and inflammatory media (vertical lines represent media alterations). 0 20 40 60 80 100 120 140 020 40 60 80 100 120 140 160 180 Oxaprozin release (%) Time (min.) Non-funtionalized NLCs + Oxa Functionalized NLCs + Oxa 0 20 40 60 80 100 120 140 020 40 60 80 100 120 140 160 180 Oxaprozin release (%) Time (min.) Non-functionalized NLCs + Oxa Functionalized NLCs + Oxa Figure 8: Oxaprozin release in gastric, intestinal and physiologic media (vertical lines represent media alterations). 41 Analysing the data (Figure 8, 9 and 10) is possible to observe that for every condition the release seems to be identical for both functionalized and non-functionalized particles revealing that the functionalization process does not interfere with the in vitro release profile. Non-functionalized oxaprozin loaded NLCs in physiological medium escape this tendency, however is also evident that the profile is unstable and the values reveal amounts of released oxaprozin superiors to those used, whereupon it is necessary to repeat the experiment to take further conclusions. As desired only a small amount of oxaprozin is released in gastric medium, less than 20%, which may help reduce oxaprozin side effects on the gastric mucosa. Most of the encapsulated oxaprozin is released on the intestinal medium leaving only about 20% of the encapsulated oxaprozin to be released on physiologic and inflammatory media. The oxaprozin released on the intestine will be absorbed by the mucosa and distributed throughout the body similarly to the free oxaprozin traditional administration. These results also indicate that the functionalization may be unnecessary or at least will play a secondary role since most of the encapsulate oxaprozin is released on the intestinal medium leaving only about 20% of the encapsulated oxaprozin to be delivery through macrophage targeting on inflamed regions. One can also verify that approximately 10% of the encapsulated oxaprozin remains entrapped on the crystalline matrix of the lipid nanoparticles. 3.5 - Effect of LNs on cell viability and cytotoxicity In order to evaluate the cytotoxicity of the designed nanoparticles in the intestinal mucosa MTT and LDH assays were performed using Caco-2 cell line (Figure 11). On both tests the assayed values of cells incubated with the nanoparticles with different oxaprozin Figure 10: Oxaprozin release in gastric, intestinal, physiological and inflammatory media (vertical lines represent media alterations). 0 10 20 30 40 50 60 70 80 90 100 020 40 60 80 100 120 140 160 180 Oxaprozin release (%) Time (min.) Non-functionalized NLCs + Oxa Functionalized NLCs + Oxa 48 free oxaprozin and a similar apparent permeability both in PBS and simulated fasted intestinal fluid. The designed particles and the obtained results suggest that this formulation enclose a huge potential for oxaprozin oral administration with potentially less significant gastric side effects, since the great majority of the entrapped oxaprozin is only released on the intestine, avoiding this way its contact with the gastric mucosa and its negative effects on it. The functionalization process seems to not interfere with the particles characteristics however since oxaprozin is mainly released in the intestine its role is secondary because only a small amount of oxaprozin remains on the particles to be released on the inflamed areas. Despite the obtained results further tests and optimization of these particles are still needed and conducted until these nanosystems become a therapeutic reality. 49 Chapter 5 Future work For future work, lipid nanoparticles functionalization yield needs to be assessed in order to evaluate the efficiency of the functionalization process which can be achieved by nuclear Magnetic Resonance (NMR) spectroscopy. Stability studies along the time need also to be conducted in order to define the LNs shelf-time. It is also important to define a lyophilisation protocol that does not compromise the LNs characteristics. Cytotoxic studies must also be done in different cell lines (gastric, cardiac and macrophages) in order to verify that the LNs effectively reduce oxaprozin cytotoxicity and consequently its side effects. 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