High-grade extracellular vesicles preparation by combined size-exclusion and affinity chromatography

Extracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of EVs such as exosomes and ectosomes, which are involved in distinct biological processes. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs

Enhanced Nanoencapsulation of Sepiapterin within PEG-PCL Nanoparticles by Complexation with Triacetyl-Beta Cyclodextrin

In this work, we aimed to improve the encapsulation efficiency of sepiapterin (SP), the natural precursor of the essential cofactor tetrahydrobiopterin (BH4) that displays mild water-solubility and a short biological half-life, within methoxy-poly(ethylene-glycol)-poly(epsilon-caprolactone)(mPEG-PCL) nanoparticles (NPs) by means of its complexation and hydrophobization with 2,3,6-triacetyl-β-cyclodextrin (TAβCD). For this, SP/TAβCD complexes were produced by spray-drying of SP/TAβCD binary solutions in ethanol using the Nano Spray Dryer B-90 HP. Dry powders were characterized by differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), and transmission and scanning electron microscopy (TEM and SEM, respectively) and compared to the pristine components and their physical mixtures (PMs). Next, SP was encapsulated within mPEG-PCL NPs by nano-precipitation of an SP/TAβCD complex/mPEG-PCL solution. In addition to the nano-encapsulation of a preformed complex within the polymeric NPs, we assessed an alternative encapsulation approach called drying with copolymer (DWC) in which pristine SP, TAβCD, and mPEG-PCL were co-dissolved in a mixture of acetone and methanol at the desired weight ratio, dried under vacuum, re-dissolved, and nano-precipitated in water. The dissolution-drying step was aimed to promote the formation of molecular hydrophobic interactions between SP, TAβCD, and the PCL blocks in the copolymer. SP-loaded mPEG-PCL NPs were characterized by dynamic light scattering (DLS) and SEM. NPs with a size of 74–75 nm and standard deviation (S.D., a measure of the peak width) of 21–22 nm were obtained when an SP:TAβCD (1:1 molar ratio) spray-dried complex was used for the nano-encapsulation and SEM analysis revealed the absence of free SP crystals. The encapsulation efficiency (%EE) and drug loading (%DL) were 85% and 2.6%, respectively, as opposed to the much lower values (14% and 0.6%, respectively) achieved with pristine SP. Moreover, the NPs sustained the SP release with relatively low burst effect of 20%. Overall, our results confirmed that spray-drying of SP/TAβCD solutions at the appropriate molar ratio leads to the hydrophobization of the relatively hydrophilic SP molecule, enabling its encapsulation within mPEG-PCL NPs and paves the way for the use of this strategy in the development of novel drug delivery systems of this vital biological precursor

Folate Receptor α-Modified Nanoparticles for Targeting of the Central Nervous System

Effective and timely delivery of therapeutic agents from the systemic circulation to the central nervous system (CNS) is often precluded by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). A new pathway of folate uptake mediated by folate receptor alpha (FRα, molecular weight of 28.29 kg mol$^{-1}$) occurring in various epithelial cells of the CNS (e.g., choroid plexus) was described. Aiming to investigate this mechanism for the delivery of nanomedicines to the CNS, in this work, we initially produced nanoparticles (NPs) made of a highly hydrophobic poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) block copolymer functionalized with an amine moiety in the edge of the PEG block by a simple nanoprecipitation method. Hydrophilic PEG blocks migrated to the NP surface during formation, exposing primary amine groups that were used to conjugate the targeting ligand, FRα. The size of the NPs was in the 58-98 nm range and standard deviation (S.D., a measure of the size population peak width) of 26-41 nm, as measured by dynamic light scattering (DLS). The FRα conjugation yield ranged between 50% and 75% (determined indirectly by the bicinchoninic acid protein assay). Pristine and FRα-modified NPs showed good compatibility with primary human choroid plexus epithelial cells (HCPEpiCs). The uptake of FRα-conjugated NPs by HCPEpiCs was qualitatively evaluated in vitro using inverted optical fluorescence and confocal microscopy. FRα-modified NPs were internalized by HCPEpiCs to a greater extent than the unmodified counterparts. Then, their permeability was characterized in standard and inverted HCPEpiC monolayers. In both cases, NPs surface modified with the FRα and complexed to folic acid (FA) showed significantly higher apparent permeability coefficient (P$_{app}$) values than the pristine ones. Finally, the biodistribution of unmodified and FRα-FA-modified NPs following intravenous (i.v.) administration was compared in ICR mice. Results indicated that conjugation of the FRα-FA complex to the NP surface promotes higher accumulation in the brain, highlighting the promise of FRα-FA-modified NPs to serve as a platform for the targeting of active molecules to the CNS from the systemic circulation

Combination of hyaluronic acid and PLGA particles as hybrid systems for viscosupplementation in osteoarthritis

Hyaluronic acid (HA) is commonly used through intra-articular administration for viscosupplementation in osteoarthritis and other disorders. HA is generally supplied as an injection commonly reported as painful, with strong limitations after treatment. In this study, an alternative delivery system was constructed based on HA hydrogel and poly(lactic-co-glycolic acid) (PLGA) particles with oleic acid. Development studies included the determination of particle toxicity, hemolytic activity, in vitro and in vivo anti-inflammatory activity using macrophages and a murine model, respectively. This study showed that empty PLGA particles presented a mean size of 373 nm, while particles containing HA and oleic acid showed a marked particle size increase. The HA association efficiency was of 73.6% and 86.2% for PLGA particles without and with oleic acid, respectively. The in vitro HA release from PLGA particles revealed a sustained profile. Particles showed a good in vitro cell compatibility and the risk of hemolysis was less < 1%, ensuring their safety. The in vivo anti-inflammatory study showed a higher inhibition for HA-loaded PLGA particles when compared to HA solution (78% versus 60%) and they were not different from the positive control, clearly suggesting that this formulation may be a promising alternative to the current HA commercial dosage form.iMed.ULisboa [UID/DTP/04138/2013]CESAM [UID/AMB/50017/2019]FCT/MEC through Portuguese fundsFEDER, within the PT2020 Partnership AgreementCompete 202

Targeted drug distribution in tumor extracellular fluid of GD2-expressing neuroblastoma patient-derived xenografts using SN-38-loaded nanoparticles conjugated to the monoclonal antibody 3F8

Neuroblastoma is a pediatric solid tumor with high expression of the tumor associated antigen disialoganglioside GD2. Despite initial response to induction therapy, nearly 50% of high-risk neuroblastomas recur because of chemoresistance. Here we encapsulated the topoisomerase-I inhibitor SN-38 in polymeric nanoparticles (NPs) surface-decorated with the anti-GD2 mouse mAb 3F8 at a mean density of seven antibody molecules per NP. The accumulation of drug-loaded NPs targeted with 3F8 versus with control antibody was monitored by microdialysis in patient-derived GD2-expressing neuroblastoma xenografts. We showed that the extent of tumor penetration by SN-38 was significantly higher in mice receiving the targeted nano-drug delivery system when compared to non-targeted system or free drug. This selective penetration of the tumor extracellular fluid translated into a strong anti-tumor effect prolonging survival of mice bearing GD2-high neuroblastomas in vivo.Fil: Monterrubio, Carles. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Paco, Sonia. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Olaciregui, Nagore G.. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Pascual Pasto, Guillem. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Vila Ubach, Monica. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Cuadrado Vilanova, Maria. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Ferrandiz, M. Mar. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Castillo Ecija, Helena. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Glisoni, Romina Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Kuplennik, Nataliya. Technion-Israel Institute of Technology; IsraelFil: Jungbluth, Achim. Memorial Sloan-Kettering Cancer Center; Estados UnidosFil: de Torres, Carmen. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Lavarino, Cinzia. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Cheung, N. K. V.. Memorial Sloan-Kettering Cancer Center; Estados UnidosFil: Mora, Jaume. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Sosnik, Alejandro Dario. Technion-Israel Institute of Technology; IsraelFil: Montero Carcaboso, Angel. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; Españ