Soft nanocarrier development as a versatile approach to brain delivery and targeting

The blood–brain barrier (BBB) is a selective system of endothelial cells, joined through the tight junctions, that protect and maintain the homeostasis of the Central Nervous System (CNS). Only small molecule or drugs, with high lipid solubility and a lower molecular mass under 400–500 Da, can cross the BBB in pharmacologically significant amounts, limiting the uptake of most therapeutic agents into the brain. Currently, the management of neurological with aggressive and invasive techniques (surgery, see Appendix 3), achieves higher therapeutic effect compared to conventional delivery methods, such as systemic administration via intravenous injection or oral administration. However invasive techniques achieve less compliance. This has led to the development of vogue treatments such as nose-to-brain technologies (see Appendix 2), FUS-induced BBB opening, fusion protein chaperones, stem cells, gene therapy, use of natural compounds and neuroprotectants, in order to increase the neuroavailability of various advanced drug delivery systems. These strategies provide promising alternatives that are able to ameliorate the treatment of brain disease. At this purpose, several nanocarriers ranging from the more established systems, e.g. polymeric nanoparticles, liposomes, niosomes and micelles to the newer systems, e.g. nanoemulsions, micro and nano-bubbles, nanosuspensions and nanogels, have been studied for the delivery of therapeutics to CNS. Many of the proposed nanomedicines can be effectively transported across various in vitro and in vivo BBB models by endocytosis and/or transcytosis, and demonstrated early preclinical success for the management of CNS conditions such as brain tumours, HIV encephalopathy, neurodegenerative disease and acute ischemic stroke. Future development of CNS nanomedicines need to focus on increasing their drug-trafficking performance and specificity for brain tissue using novel targeting moieties, improving their BBB permeability and reducing their neurotoxicity. Targeted drug delivery is a means of concentrating drugs at a specific site relative to other parts of the body. It spares the rest of the body from toxic effects of the drug and it is also a potential means of improving therapeutic index. This Ph.D. thesis focused on the study of the formulation of different nanocarriers for brain delivery by two different administration routes: (1) Focused Ultrasound-mediated drug delivery, a technique that offer a unique non-invasive avenue to deliver drugs to the brain through transient opening of the BBB by using of ultrasound in combination with gas-filled bubbles. Our laboratory developed new carriers, the “Bubblesomes”®, able to combine the characteristics of a drug carrier and a contrast agent (theranostic system). When focused ultrasound is applied in presence of drug loaded nanobubbles intravenously administered, inertial cavitation is induced, due to the rapid expansion and violent collapsing of bubbles. This can cause the temporal and fully reversible opening of BBB due to the enhanced endothelial porosity and vascular permeability phenomenon called sonoporation, resulting in an increased drug uptake. (2) Intranasal drug delivery, a non-invasive route to reach directly the brain, circumvent the BBB, from the nose along the olfactory and trigeminal nerve pathways. These nerve pathways initiate in the nasal cavity at olfactory neuroepithelium and terminate in the brain

Drug delivery in overcoming the blood-brain barrier: role of nasal mucosal grafting

The blood–brain barrier (BBB) plays a fundamental role in protecting and maintaining the homeostasis of the brain. For this reason, drug delivery to the brain is much more difficult than that to other compartments of the body. In order to bypass or cross the BBB, many strategies have been developed: invasive techniques, such as temporary disruption of the BBB or direct intraventricular and intracerebral administration of the drug, as well as noninvasive techniques. Preliminary results, reported in the large number of studies on the potential strategies for brain delivery, are encouraging, but it is far too early to draw any conclusion about the actual use of these therapeutic approaches. Among the most recent, but still pioneering, approaches related to the nasal mucosa properties, the permeabilization of the BBB via nasal mucosal engrafting can offer new potential opportunities. It should be emphasized that this surgical procedure is quite invasive, but the implication for patient outcome needs to be compared to the gold standard of direct intracranial injection, and evaluated whilst keeping in mind that central nervous system diseases and lysosomal storage diseases are chronic and severely debilitating and that up to now no therapy seems to be completely successful

Neem oil nanoemulsions: characterisation and antioxidant activity

The aim of the present work is to develop nanoemulsions (NEs), nanosized emulsions, manufactured for improving the delivery of active pharmaceutical ingredients. In particular, nanoemulsions composed of Neem seed oil, contain rich bioactive components, and Tween 20 as nonionic surfactant were prepared. A mean droplet size ranging from 10 to 100nm was obtained by modulating the oil/surfactant ratio. Physicochemical characterisation was carried out evaluating size, f-potential, microviscosity, polarity and turbidity of the external shell and morphology, along with stability in simulated cerebrospinal fluid (CSF), activity of Neem oil alone and in NEs, HEp-2 cell interaction and cytotoxicity studies. This study confirms the formation of NEs by Tween 20 and Neem oil at different weight ratios with small and homogenous dimensions. The antioxidant activity of Neem oil alone and in NEs was comparable, whereas its cytotoxicity was strongly reduced when loaded in NEs after interaction with HEp-2 cells

Rifampicin-liposomes for mycobacterium abscessus infection treatment: intracellular uptake and antibacterial activity evaluation

: Treatment of pulmonary infections caused by Mycobacterium abscessus are extremely difficult to treat, as this species is naturally resistant to many common antibiotics. Liposomes are vesicular nanocarriers suitable for hydrophilic and lipophilic drug loading, able to deliver drugs to the target site, and successfully used in different pharmaceutical applications. Moreover, liposomes are biocompatible, biodegradable and nontoxic vesicles and nebulized liposomes are efficient in targeting antibacterial agents to macrophages. The present aim was to formulate rifampicin-loaded liposomes (RIF-Lipo) for lung delivery, in order to increase the local concentration of the antibiotic. Unilamellar liposomal vesicles composed of anionic DPPG mixed with HSPC for rifampicin delivery were designed, prepared, and characterized. Samples were prepared by using the thin-film hydration method. RIF-Lipo and unloaded liposomes were characterized in terms of size, ζ-potential, bilayer features, stability and in different biological media. Rifampicin's entrapment efficiency and release were also evaluated. Finally, biological activity of RIF-loaded liposomes in Mycobacterium abscessus-infected macrophages was investigated. The results show that RIF-lipo induce a significantly better reduction of intracellular Mycobacterium abscessus viability than the treatment with free drug. Liposome formulation of rifampicin may represent a valuable strategy to enhance the biological activity of the drug against intracellular mycobacteria

Ultrastable shelled PFC nanobubbles:a platform for ultrasound-assisted diagnostics and therapy

Nanoscale echogenic bubbles (NBs), can be used as a theranostic platform for the localized delivery of encapsulated drugs. However, the generation of NBs is challenging, because they have lifetimes as short as milliseconds in solution. The aim of this work has been the optimization of a preparation method for the generation of stable NBs, characterized by measuring: a) acoustic efficiency, b) nano-size, to ensure passive tumour targeting, c) stability during storage and after injection and d) ability to entrap drugs. NBs are monodisperse and ultrastable, their stability achieved by generation of an amphiphilic multilamellar shell able to efficiently retain the PFC gas. The NBs perform as good acoustic enhancers over a wide frequency range and out of resonant conditions, as tested in both in vitro and in vivo experiments, proving to be a potential platform for the production of versatile carriers to be used in ultrasound-assisted diagnostic, therapeutic and theranostic applications

Effect of ciprofloxacin-loaded niosomes on escherichia coli and staphylococcus aureus biofilm formation

Infections caused by bacterial biofilms represent a global health problem, causing considerable patient morbidity and mortality in addition to an economic burden. Escherichia coli, Staphylococcus aureus, and other medically relevant bacterial strains colonize clinical surfaces and medical devices via biofilm in which bacterial cells are protected from the action of the immune system, disinfectants, and antibiotics. Several approaches have been investigated to inhibit and disperse bacterial biofilms, and the use of drug delivery could represent a fascinating strategy. Ciprofloxacin (CIP), which belongs to the class of fluoroquinolones, has been extensively used against various bacterial infections, and its loading in nanocarriers, such as niosomes, could support the CIP antibiofilm activity. Niosomes, composed of two surfactants (Tween 85 and Span 80) without the presence of cholesterol, are prepared and characterized considering the following features: hydrodynamic diameter, ζ-potential, morphology, vesicle bilayer characteristics, physical-chemical stability, and biological efficacy. The obtained results suggest that: (i) niosomes by surfactants in the absence of cholesterol are formed, can entrap CIP, and are stable over time and in artificial biological media; (ii) the CIP inclusion in nanocarriers increase its stability, with respect to free drug; (iii) niosomes preparations were able to induce a relevant inhibition of biofilm formation

Neem oil or almond oil nanoemulsions for vitamin E Delivery: from structural evaluation to in vivo assessment of antioxidant and anti-inflammatory activity

Purpose: Vitamin E (VitE) may be classified in "the first line of defense" against the formation of reactive oxygen species. Its inclusion in nanoemulsions (NEs) is a promising alternative to increase its bioavailability. The aim of this study was to compare O/W NEs including VitE based on Almond or Neem oil, showing themselves antioxidant properties. The potential synergy of the antioxidant activities of oils and vitamin E, co-formulated in NEs, was explored. Patients and methods: NEs have been prepared by sonication and deeply characterized evaluating size, ζ-potential, morphology (TEM and SAXS analyses), oil nanodroplet feature, and stability. Antioxidant activity has been evaluated in vitro, in non-tumorigenic HaCaT keratinocytes, and in vivo through fluorescence analysis of C. elegans transgenic strain. Moreover, on healthy human volunteers, skin tolerability and anti-inflammatory activity were evaluated by measuring the reduction of the skin erythema induced by the application of a skin chemical irritant (methyl-nicotinate). Results: Results confirm that Vitamin E can be formulated in highly stable NEs showing good antioxidant activity on keratinocyte and on C. elegans. Interestingly, only Neem oil NEs showed some anti-inflammatory activity on healthy volunteers. Conclusion: From the obtained results, Neem over Almond oil is a more appropriate candidate for further studies on this application

DLS Characterization of Non-Ionic Surfactant Vesicles for Potential Nose to Brain Application

The aim of this paper is the preparation and characterization of drug delivery systems for a potential brain delivery by intranasal administration. It is possible to reach the central nervous system with alternative routes through which therapeutic agents can bypass the blood brain barrier: that is the nasal administration. Intranasal drug administration is non-invasive and it could be a promising drug delivery method for patients who suffer from chronic and crippling Central Nervous System diseases. Among the formulation strategies for enhanced nose to brain drug delivery, the use of colloidal carriers has became a revolutionary approach. The success of a therapeutic strategy by using nanocarriers depends on their ability to entrap drugs, to penetrate through anatomical barriers, to efficiently release the incorporated drugs, to show a good stability in nanometric size range and good biocompatibility. The use of vesicular systems (niosomes), in nose to brain delivery is here presented. One of the major problems associated with nasal administration is the rapid removal of drugs or drug delivery systems, from the deposition site through mucociliary clearance. This effect is responsible of reduction of contact time between drug or drug delivery systems and nasal epithelium. This problem could be solved by coating nanocarriers with a mucoadhesive agent: chitosan. In this paper the preparation and characterization of hybrid niosomes by Tween 20 and Tween 21 together with dicetyl phosphate or Span 20 and the cationic polyelectrolyte chitosan are described in order to obtain intranasal drug delivery systems. In particular through dynamic light scattering, laser Doppler electrophoresis and fluorescence measurements the aggregation behavior between vesicles and polyelectrolyte can be monitored. Overall phenomenology is well described in terms of the re-entrant condensation and charge inversion behavior, observed in different colloidal systems. The physical stability of hybrid niosomes obtained by the three different surfactants was also evaluated

LIPOSOMAL APPROACH TO PHARMACEUTICS

Efficient and safe drug delivery has always been a challenge in therapy and diagnostics. The use of nanotechnology, as well as the development of nanocarriers for drug delivery, received great attention according to the evidence that they can theoretically act as “magic bullets” capable to hit the target cells while sparing normal tissues and organs. Liposomes are suitable as carriers of both hydrophilic and lipophilic drugs and are able to deliver drugs to the target site. The vesicle properties are specifically dictated by size, shape, and surface chemistry which are able to modify the intrinsic pharmacokinetics of the drug and, eventually, the drug targeting to the pathological areas. Liposomes can be developed in different approach to pharmaceutics. According to this, in our laboratory, liposomes have been proposed as biomembrane model in order to study the interaction between surfactant vesicles and liposomes, since it is very difficult to directly observe the membrane fusion due to the complexity of biomembranes and the high speed of the process (1). Furthermore our research involves the “classic” use of liposomes as drug delivery systems. For example they have been studied to obtain an efficient brain delivery of prodrugs to enhance the extracellular levels of L-Dopa and Dopamine in rat striatum of freely moving rat (2). Maleic and fumaric diamides of (O,O-diacetyl)- L-Dopa-methylester, synthesized in order to attenuate marked fluctuations of L-Dopa plasma levels and to overcome the problem of low bioavailability of L-Dopa, have been entrapped in liposomal formulations to obtain chemical stability in aqueous buffer solutions, slow release of LD in human plasma and sustained delivery of Dopamine in rat striatal dialysate (3). With the aim to provide a protection against chemical degradation and enzymatic metabolism and, consequently, to get a high prodrug quantity able to cross the Blood Brain Barrier, liposomal formulations of 2-amino-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (DOPH) were prepared and characterized (4). Polysaccharide-coated liposomes have been proposed as carrier of peptide drug by the oral route because they are able to minimize the disruptive influences on peptide drugs of gastrointestinal fluids. In particular, a modified polysaccharide, O-palmitoylscleroglucan (PSCG), was synthesized and used to coat unilamellar liposomes for oral delivery of Leuprolide, a synthetic superpotent agonist of luteinizing hormone releasing hormone receptor (5). In collaboration with the Centre for Surgical Technologies at the Faculty of Medicine of the Katholieke Universiteit of Leuven (Belgium), thermosensitive liposomes have been prepared to deliver the Cytotoxic Necrotizing Factor 1 (CNF-1) toward lung tissues, with an effective approach against the Congenital Diaphragmatic Hernia (CDH). These carriers are able to release drugs/proteins due to the hyperthermia, as a consequence of lung tissues inflammation. The preliminary studies have shown the safety of these structures in foetal rats and rabbits with CDH. Finally, in our laboratory, the phospholipidic shell was used to prepare novel nanobbubles, NBs (6) - ). These structures can act as theranostic agents. The preclinical experiments, developed in collaboration with the Queens Medical Research Institute at the University of Edinburgh, were carried out to demonstrate that ultrasound-mediated NBs destruction has the potential to open the BBB tight junctions and trigger therapeutic agent deposition in the brain. The results are very promising and suggest the possible use of NBs in the theranostic fields