Highly specific blood-brain barrier transmigrating single-domain antibodies selected by an In Vivo phage display screening

Research Areas: Pharmacology & PharmacyA major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.info:eu-repo/semantics/publishedVersio

Nano-and microcarriers as drug delivery systems for usnic acid: Review of literature

Usnic acid is one of the most investigated lichen secondary metabolites, with several proven biological properties with potential medical relevance. However, its unfavorable physico-chemical properties, as well as observed hepatotoxicity, have discouraged wide-range utilization of usnic acid as a promising therapeutic agent. In accordance with the growing research interest in the development of nanotechnology, especially in the arena of preparations based on natural sources of medicinal compounds, usnic acid incorporated into nano-and microsized colloidal carriers has been a subject of a large number of publications. Therefore, this review discusses the overall results of the studies dealing with usnic acid encapsulated into lipid-based, polymeric and nonorganic micro-and/or nanocarriers, as potential drug delivery systems for this natural compound, in an attempt to introduce its usage as a potential antitumor, antimicrobial, wound-healing, antioxidative and anti-inflammatory drug

Pulmonary delivery of antibiotic-loaded lipid nanoparticles for the treatment of Pseudomonas aeruginosa infections associated with cystic fibrosis

225 p.El objetivo principal de esta tesis doctoral es el desarrollo de nanopartículas lipídicas sólidas (SLN) y partículas lipídicas nanoestructuradas (NLC) como vehículos para facilitar la liberación de antibióticos, colistimetato sódico y tobramicina, tras su administración pulmonar para el tratamiento de la fibrosis quística. La primera etapa de este trabajo fue el desarrollo y optimización de nanopartículas lipídicas cargadas con colistimetato sódico (Colist-SLN y Colist-NLC) que mostraron actividad antimicrobiana contra cepas de Pseudomonas aeruginosa aisladas de pacientes con fibrosis quística. Tras realizar un estudio de biodistribución de las NLCs en ratones usando una torre de nebulización se observó una elevada retención en los pulmones hasta las 48 horas. En una segunda parte, se realizó un estudio de estabilidad de las partículas de acuerdo con la normativa ICH. Para ello se analizaron sus características físico-químicas (tamaño, PDI, potencial zeta, perfil de liberación) y su actividad antimicrobiana durante un año. Las Colist-NLCs presentaron una mejor estabilidad a 5¿C y 25¿C/60% de humedad relativa durante un año de almacenamiento frente a las SLNs. Por ello, en la última parte de este trabajo se optó por encapsular tobramicina en NLCs (Tb-NLCs), nanopartículas que presentaron actividad antibacteriana frente a P. aeruginosa y permanecieron en los pulmones de los ratones hasta las 48 horas después de su administración intratraqueal. En resumen, teniendo en cuenta los resultados de esta tesis se puede concluir que las NLCs, utilizadas como vehículo de antibióticos, parecen una alternativa prometedora para el tratamiento de la fibrosis quísticaNanoBioCe

PEPTIDE-POLYMER CONJUGATES AS TOOLS TO SELECTIVELY TARGET EGFR

Nanoparticles (NPs) made of biodegradable and biocompatible polymers present several advantages as carriers for therapeutics. Targeted polymeric NPs, able to hit specific tissues and cells, can be obtained by synthesis of hybrid or biointegrated nanosystems where the combination of polymers with biomolecules such as peptides, proteins, or monoclonal antibodies offers opportunities to design precise and versatile nanoscale systems. The central challenge towards these \u201csmart\u201d materials is represented by the optimal interplay of biophysicochemical parameters that confer molecular targeting, immune evasion, and optimal drug release, and allow to overcome the physiological barriers in vivo. The epidermal growth factor receptor (EGFR) is a cell-surface receptor of extracellular protein ligands of the epidermal growth factor family. Mutations that lead to EGFR overexpression or overactivity have been associated with a wide spectrum of human cancers of epithelial origin, including breast and colorectal cancers, and with autoimmune disorders like rheumatoid arthritis. Recently, several studies have reported the successful identification, by screening phage display libraries, of a peptide ligand, named GE11 (YHWYGYTPQNVI), with high binding capacity to EGFR but with low mitogenic activity. Poly(gamma-glutamic acid) (gamma-PGA) is an extracellular bacterial water-soluble polymer with variable molecular weight produced by several members of the genus Bacillus, composed of D and/or L-glutamic acid monomers, connected by amide bonds between alpha-amino and gamma-carboxyl groups. gamma-PGA is readily biodegraded by a good number of bacteria, it is non-immunogenic and completely innocuous and so it seems to meet most of the requirements of polymers for drug delivery. In addition, it bears pendant carboxylic groups in \u3b1-position which are available for chemical derivatization allowing the modification of its molecular properties or the attachment of biologically active molecules. The use of gamma-PGA, however, presents some inherent and not negligible issues. First of all, chemical modification of the material is very arduous: this is a consequence of its structure but also of its scarce solubility in most organic solvents. Consequently, studies are necessar to find a feasible and efficient way to exploit it for drug delivery purposes. This PhD work aimed to find a way to valorize gamma-PGA peculiar characteristics in drug delivery field, particularly in the preparation of GE11 directed nanocarriers. We operated on two different sides: on one hand we performed intense studies on the biopolymer exploring its chemical-physical properties (mainly MW and solubility), its structure and its reactivity. On the other hand we studied and optimized methods to functionalize a well known and widely used biopolymer, poly lactic-co-glycolic acid (PLGA), with peptides. Starting from these conjugates we also prepared and characterized nanoparticles intended to be used as a drug delivery tools to EGFR overexpressing cells

TARGETED DELIVERY OF ANTICANCER AGENTS TO CANCER

Our research focuses on developing dual functional polymeric micelles for the targeted delivery of anticancer agents to tumors. We first developed a poly(ethylene) glycol (PEG)-derivatized anticancer agent-Embelin (EB) (PEG-EB2) as an effective nanomicellar carrier for the delivery of Paclitaxel (PTX) to tumors. Our data demonstrated that PEG-EB2 retained similar biological effect as EB. Surprisingly, it can self-assemble into micelles (~20 nm) in aqueous solution and was also efficient in delivering the Paclitaxel (PTX) to cancers with enhanced antitumor activity. Further, folate (FA), a tumor specific ligand, was anchored into PEG5K-EB2 micelles (FA-PEG5K-EB2) to realize the active tumor targeting. The intracellular uptake of Doxorubicin (DOX) was markedly improved when incorporated into FA-PEG5K-EB2 over the one without FA, resulting in the significant higher level in inhibiting tumor growth. Moreover, structure activity relationship (SAR) study was performed in PEG-derivatized Vitamin E (PEG-VE), in which our data has shown that PEG-VE with longer PEG length (5K) and double VE chains (PEG5K-VE2) garnered significant better PTX loading, stability and improved antitumor efficacy. Additionally, aiming to improve the DOX loading and stability, a drug-interactive motif-Fmoc was placed in the interfacial region of the PEG5K-VE2 (PEG5K-Fmoc-VE2). The data suggested that introduction of Fmoc to PEG5K-VE2 brought about dramatic augmentation in DOX loading and formulation stability, which consequently led to an enhanced inhibition on tumor development. Another finding in my research is to formulate Camptothecin (CPT), a highly lipophilic antineoplastic drug, in an innovative fashion. CPT was conjugated with VE at its hydroxyl group via carbonate ester bond (CPT-VE) or disulfide linkage (CPT-S-S-VE), which can self-assemble into nanofiber upon stabilized by PEG5K-Fmoc-VE2. VE-derivatized CPT prodrugs significantly buttressed the stability of CPT due to the additional steric hindrance to the lactone ring on CPT. Meanwhile, compared to CPT-VE, CPT-S-S-VE can more readily liberate CPT at tumors in a controlled manner (high GSH conc. in tumor), leading to the superior tumor growth suppression in vivo. To reiterate, our data demonstrated that PEG-derivatized anticancer agents can serve as effective nanocarriers for the targeted delivery of chemotherapeutics. Additionally, incorporation of Fmoc into the interfacial region of dual functional carriers led to significantly increased drug loading and formulation stability, resulting in improved antitumor activity. Furthermore, coupling of VE to anticancer drugs may represent a novel platform in ameliorating their compatibility with utilized carrier

Drug metabolism: Comparison of biodistribution profile of holmium in three different compositions in healthy Wistar rats

Radioisotope holmium is a candidate to be used in cancer treatment and diagnosis. There are different holmium salts and they present distinct solubility and consequently different biodistribution profiles. In this work, we aimed to evaluate the biodistribution profiles of two holmium salts (chloride and sulfate) and holmium nanoparticles (oxide) through an in vivo biodistribution assay using animal model. Samples were labeled with technetium-99m and administered in Wistar rats by retro-orbital route. Holmium chloride is highly soluble in water and it was quickly filtered by the kidneys while holmium sulfate that presents lower solubility in water was mainly found in the liver and the spleen. However, both the salts showed a similar biodistribution profile. On the other hand, holmium oxide showed a very different biodistribution profile since it seemed to interact with all organs. Due to its particle size range (approximately 100 nm) it was not intensively filtered by the kidneys being found in high quantities in many organs, for this reason its use as a nanoradiopharmaceutical could be promising in the oncology field. (C) 2016 Elsevier Ltd. All rights reserved.The authors would like to thank the National Scientific and Technological Research Council (CNPQ) (CNPq: 303594/2015-2) and the Rio de Janeiro State Research Foundation (FAPERJ) for funding.Cerqueira-Coutinho, C.; Pascual Vidal, L.; Pinto, SR.; Santos-Oliveira, R. (2016). Drug metabolism: Comparison of biodistribution profile of holmium in three different compositions in healthy Wistar rats. Applied Radiation and Isotopes. 112:27-30. https://doi.org/10.1016/j.apradiso.2016.02.015S273011