Lucia Bondioli

NIR-labeled nanoparticles engineered for brain targeting: in vivo optical imaging application and fluorescent microscopy evidences

The presence of the blood–brain barrier (BBB) makes extremely difficult to develop efficacious strategies for targeting contrast agents and delivering drugs inside the Central Nervous System (CNS). To overcome this drawback, several kinds of CNS-targeted nanoparticles (NPs) have been developed. In particular, we proposed poly-lactide-co-glycolide (PLGA) NPs engineered with a simil-opioid glycopeptide (g7), which have already proved to be a promising tool for achieving a successful brain targeting after i.v. administration in rats. In order to obtain CNS-targeted NPs to use for in vivo imaging, we synthesized and administrated in mice PLGA NPs with double coverage: near-infrared (NIR) probe (DY-675) and g7. The optical imaging clearly showed a brain localization of these novel NPs. Thus, a novel kind of NIR-labeled NPs were obtained, providing a new, in vivo detectable nanotechnology tool. Besides, the confocal and fluorescence microscopy evidences allowed to further confirm the ability of g7 to promote not only the rat, but also the mouse BBB crossing.

Sialic acid and glycopeptides conjugated PLGA nanoparticles for central nervous system targeting: In vivo pharmacological evidence and biodistribution

Polymeric nanoparticles (Np) have been considered as strategic carriers for brain targeting. Specific ligands on the surface allowed the Np to cross the Blood-Brain Barrier (BBB) carrying model drugs within the brain district after their i.v. administration in experimental animals. It is known that sialic acid receptors are present in several organs, including in the brain parenchyma. Thus, in this paper, we prepared PLGA Np surface modified with a BBB-penetrating peptide (similopioid peptide) for BBB crossing and with a sialic acid residue (SA) for the interaction with brain receptors. This double coverage could allow to obtain novel targeted Np with a prolonged residence within the brain parenchyma, thus letting to reach a long-lasting brain delivery of drugs. The central analgesic activity of Loperamide (opioid drug, unable to cross the BBB) loaded in these novel Np was evaluated in order to point out the capability of the Np to reach and to remain in the brain. The results showed that the pharmacological effect induced by loaded Np administration remained significant over 24h. Using confocal and fluorescent microscopies, the novel Np were localized within the tissue parenchyma (brain, kidney, liver, spleen and lung). Finally, the biodistribution studies showed a localization of the 6% of the injected dose into the CNS over a prolonged time (24h). Notwithstanding an increased accumulation of SA-covered Np in those organs showing SA-receptors (liver, kidney, and lung), the pharmacological and biodistribution results are proofs of the ability of double targeted Np to enter the brain allowing the drug to be released over a prolonged time.

AFM, ESEM, TEM, and CLSM in liposomal characterization: a comparative study

An outstanding aspect of pharmaceutical nanotechnology lies in the characterization of nanocarriers for targeting of drugs and other bioactive agents. The development of microscopic techniques has made the study of the surface and systems architecture more attractive. In the field of pharmaceutical nanosystems, researchers have collected vital information on size, stability, and bilayer organization through the microscopic characterization of liposomes. This paper aims to compare the results obtained by atomic force microscopy, environmental scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy to point out the limits and advantages of these applications in the evaluation of vesicular systems. Besides this comparative aim, our work proposes a simple confocal laser scanning microscopy procedure to rapidly and easily detect the liposomal membrane.

Investigation on mechanisms of glycopeptide nanoparticles for drug delivery across the blood–brain barrier

AIM Nanoneuroscience, based on the use polymeric nanoparticles (NPs), represents an emerging field of research for achieving an effective therapy for neurodegenerative diseases. In particular, poly-lactide-co-glycolide (PLGA) glyco-heptapetide-conjugated NPs (g7-NPs) were shown to be able to cross the blood-brain barrier (BBB). However, the in vivo mechanisms of the BBB crossing of this kind of NP has not been investigated until now. This article aimed to develop a deep understanding of the mechanism of BBB crossing of the modified NPs. MATERIALS & METHODS Loperamide and rhodamine-123 (model drugs unable to cross the BBB) were loaded into NPs, composed of a mixture of PLGA, differently modified with g7 or with a random sequence of the same aminoamids (random-g7). To study brain targeting of these model drugs, loaded NPs were administered via the tail vein in rats in order to perform both pharmacological studies and biodistribution analysis along with fluorescent, confocal and electron microscopy analysis, in order to achieve the NP BBB crossing mechanism. Computational analysis on the conformation of the g7- and random-g7-NPs of the NP surface was also developed. RESULTS Only loperamide delivered to the brain with g7-NPs created a high central analgesia, corresponding to the 14% of the injected dose, and data were confirmed by biodistribution studies. Electron photomicrographs showed the ability of g7-NPs in crossing the BBB as evidenced by several endocytotic vesicles and macropinocytotic processes. The computational analysis on g7 and random-g7 showed a different conformation (linear vs globular), thus suggesting a different interaction with the BBB. CONCLUSION Taken together, this evidence suggested that g7-NP BBB crossing is enabled by multiple pathways, mainly membrane-membrane interaction and macropinocytosis-like mechanisms. The results of the computational analysis showed the Biousian structure of the g7 peptide, in contrast to random-g7 peptide (globular conformation), suggesting that this difference is pivotal in explaining the BBB crossing and allowing us to hypothesize regarding the mechanism of BBB crossing by g7-NPs.

PLGA nanoparticles surface decorated with the sialic acid, N-acetylneuraminic acid.

There is a broad interest in the development of nanoparticles (NPs) carrying on their surface carbohydrates such as sialic acids. It is known that these carbohydrates influence the biological and physical properties of biopharmaceutical proteins and living cells. Macromolecular compounds containing these carbohydrates showed an anti-recognition effect, exert an antiviral effect and also are able to be recognized by the cell surface of some kind of cancer cells. Thus, in the present research we performed two different approaches in order to obtain polymeric (poly(D,L-lactide-co-glycolide), PLGA) NPs surface decorated with the sialic acid N-acetylneuraminic acid (Neu5Ac). The first strategy that has been followed is based on the derivatization of the polyester PLGA with the thioderivative of Neu5Ac, starting material for the preparation of the NPs; the second is based on the synthesis of compounds potentially able to insert their lipophilic moiety into the underivatized PLGA NPs during their preparation, and to display their hydrophilic moiety (Neu5Ac) on their surface. The first approach allowed the obtainment of NPs surface decorated with Neu5Ac, as evidenced by ESCA spectroscopy and interaction with the lectin Wheat Germ Agglutinin. Moreover, a formulation of these NPs suitable for in vitro assays showed that they are phagocytosed by human monocytes with an apparently different mechanism with respect of those made of underivatized PLGA. The second strategy led to NPs in which their surface appears to be very different with respect to the NPs obtained following the first strategy, with the carboxylic groups of Neu5Ac markedly shielded. Thus, the new Neu5Ac-modified PLGA polyester represent a useful starting material for the preparation of NPs surface decorated with this sialic acid.

Development of Novel Zn2+ Loaded Nanoparticles Designed for Cell-Type Targeted Drug Release in CNS Neurons: In Vitro Evidences

Intact synaptic function and plasticity are fundamental prerequisites to a healthy brain. Therefore, synaptic proteins are one of the major targets for drugs used as neuro-chemical therapeutics. Unfortunately, the majority of drugs is not able to cross the blood–brain barrier (BBB) and is therefore distributed within the CNS parenchyma. Here, we report the development of novel biodegradable Nanoparticles (NPs), made of poly-lactide-co-glycolide (PLGA) conjugated with glycopeptides that are able to cross the BBB and deliver for example Zn2+ ions. We also provide a thorough characterization of loaded and unloaded NPs for their stability, cellular uptake, release properties, toxicity, and impact on cell trafficking. Our data reveal that these NPs are biocompatible, and can be used to elevate intracellular levels of Zn2+. Importantly, by engineering the surface of NPs with antibodies against NCAM1 and CD44, we were able to selectively target neurons or glial cells, respectively. Our results indicate that these biodegradable NPs provide a potential new venue for the delivery Zn2+ to the CNS and thus a means to explore the influence of altered zinc levels linked to neuropsychological disorders such as depression.

Can leptin-derived sequence-modified nanoparticles be suitable tools for brain delivery?

AIM In order to increase the knowledge on the use of nanoparticles (NPs) in brain targeting, this article describes the conjugation of the sequence 12-32 (g21) of leptin to poly-lactide-co-glycolide NPs. The capability of these modified NPs to reach the brain was evaluated in rats after intravenous administration. MATERIALS & METHODS The g21 was linked on the surface of NPs labeled with tetramethylrhodamine by means of the Avidin-Biotin technology. The g21-labeled NPs were injected into the tail vein of rats and, after animal sacrifice, the brain localization was evaluated by confocal microscopy, fluorescence microscopy and electron microscopy. Studies to evaluate the biodistribution of the g21-modified NPs in comparison to the unmodified NPs were also carried out. Moreover, to confirm the absence of any anorectic effect of g21 linked on the surface of NPs, appropriate studies were used to assess the rats. RESULTS After intravenous administration, the g21-modified NPs were able to cross the blood-brain barrier and to enter the brain parenchyma. The biodistribution studies of both unmodified and modified NPs pointed out an uptake at liver and spleen level, whereas only the g21-modified NPs showed brain localization. The food-intake experiments pointed out that the intravenous administration of g21 conjugated to the NP surface did not produce any anorectic effect in the rats. CONCLUSION g21-modified NPs were able to cross the blood-brain barrier. These new modified NPs could be effectively considered as useful carrier systems for brain drug delivery.

Synthesis, activity and molecular modeling of a new series of chromones as low molecular weight protein tyrosine phosphatase inhibitors.

Protein tyrosine phosphatases (PTP) are crucial elements in eukaryotic signal transduction. Several reports suggested that the LMW-PTP family has oncogenic relevance. Moreover, LMW-PTP has been recognized as a negative regulator of insulin-mediated mitotic and metabolic signaling. Thus, inhibition of the LMW-PTP can be considered an attractive approach for the design of new therapeutic agents for the treatment of type II diabetes and for new antitumoral drugs. To date very few (and weak) inhibitors of LMW-PTP have been identified. On the basis of the reported weak activity of some flavonoids on phosphatases, we discovered a lead that originated a new class of highly active LMW-PTP inhibitors; these compounds inhibit also PTP-1B and are active in cellular assays. Docking experiments and SAR highlighted the possible binding mode of these compounds to the enzyme, putting the background for the future optimization of their inhibitory activity and selectivity towards the closely related enzyme PTP-1B.

Chapter 3 - Colloidal systems for CNS drug delivery.

The pharmaceutical treatment of central nervous system (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, noninvasive drug delivery systems for CNS are actively studied. The development of these new delivery systems started with the discovery that properly surface-engineered colloidal vectors, and in particular liposomes and polymeric nanoparticles, with a diameter approximately 200nm, were shown to be able to cross the blood-brain barrier (BBB) without apparent damage, and to deliver drugs or genetic materials into the brain. However, even if this ability was confirmed by confocal microscopy and measured by biodistribution experiments or by means of the pharmacological effect exerted by the embedded drugs, a clear understanding of the main characteristics of the colloidal systems that are important for BBB crossing is still lacking. It is also shown that the presence of the drug is able to modify the surface of these systems, with unpredictable results on the colloidal systems biodistribution; thus, the results obtained in the absence of the loaded drug have to be taken cautiously. Moreover, since the loaded drug is only a fraction of the colloidal system that is administered, the presence of the carrier in the body and into CNS, especially in the case of long-term therapies, might cause adverse effects not yet fully understood. Thus, even if promising results have been obtained, and some colloidal systems loaded with a drug are the US Food and Drug Administration (FDA) approved for human use (but not for brain targeting), a long way of research has to be done in order to use these drug delivery systems for the treatment of CNS pathologies.

AFM phase imaging of soft-hydrated samples: A versatile tool to complete the chemical-physical study of liposomes

Despite of the several approaches applied to the physicochemical characterization of liposomes, few techniques are really useful to obtain information about the surface properties of these colloidal drug-delivery systems. In this paper, we demonstrate a possible new application of tapping mode atomic force microscopy (AFM) to discriminate between conventional and pegylated liposomes. We showed that the differences on liposomal surface properties revealed by the phase images AFM approach well correlate with the data obtained using classical methods, such as light scattering, hydrodynamic, and nuclear magnetic resonance analysis.