PEGylated cationic liposome - DNA nanoparticle assembly in cell culture media: pathway effects and clues to enhanced control and transfection efficiency optimization

XII Spanish-Portuguese Conference on Controlled Drug DeliveryCationic Liposome – DNA nanoparticles constitute a promising approach for safe and efficient delivery of genes for therapeutic applications. In order to be used in vivo, these particles can be coated with an inert and hydrophilic polymer, such as polyethylene-glycol (PEG), which improves blood circulation time by providing steric stabilization against removal by the immune system. In this work we study the influence of the initial salt concentration, which controls the electrostatic atraction between cationic liposomes and anionic DNA, on the structure of PEGylated CL–DNA nanoparticles.info:eu-repo/semantics/publishedVersio

Pathway-dependent effects on the formation of bioreducible polycation-DNA polyplexes in saline media

This research is supported by Microfluidic Layer-by-layer Assembly of Cationic Liposome - Nucleic Acid Nanoparticles for Gene Delivery project (032520) co-funded by FCT and the ERDF through COMPETE2020.info:eu-repo/semantics/publishedVersio

Exploring endolysin-loaded liposomes for a transtympanic treatment of S. pneumoniae otitis media

Otitis media, the main reason for which antibiotics are prescribed in childhood, is often caused by Streptococcus pneumoniae. The exogenous use of recombinantly produced endolysins, peptidoglycan hydrolases encoded by bacteriophages at the end of their lytic cycle, have been shown to be very effective against this pathogen. To increase bioavailability, and consequently reduce the probability of a recurrent or chronic infection, endolysins could be applied topically in the ear. However, delivery systems with permeation enhancing characteristics are needed to surpass the barrier provided by the tympanic membrane, which separates the ear canal from the middle ear. Therefore, this work aimed to develop a novel delivery system for a transtympanic treatment of pneumococcal otitis media using endolysins. The MSlys endolysin was encapsulated into deformable liposomes composed of L-alpha-lecithin and sodium cholate (L:SC:MSlys) or PEG2000 PE (L:PEG:MSlys) with a efficiency of approximately 35% in average, being released in a controlled manner. Liposomes loaded with MSlys showed no cytotoxicity against keratinocyte and fibroblast cell lines. Moreover, MSlys-loaded liposomes interacted with S. pneumoniae cells, being able to significantly reduce planktonic and biofilm cells. Transtympanic permeation studies demonstrated that PEGylated liposomes significantly enhanced the transport of MSlys through human tympanic membranes in an ex vivo model, showing antipneumococcal effect after 2 hours. Nevertheless, degradation of MSlys occurred during extended incubation at 37 ºC, which affected its effectiveness. In conclusion, endolysin-loaded liposomes are a promising approach for transtympanic treatment of otitis media caused by S. pneumoniae. Nevertheless, further optimization is required in order to increase effectiveness.info:eu-repo/semantics/publishedVersio

Liposomes loaded with the pneumococcal endolysin MSlys: From in vitro characterization to ex vivo permeation across the tympanic membrane

Introduction: The increasing antibiotic resistance triggered interest in novel antimicrobials as well as delivery systems that allow a topical and targeted delivery. However, biological barriers, such as the skin or the tympanic membrane (TM), may hinder the success of the therapy. Drug permeation has been extensively studied in the context of transdermal delivery, but only recently started to be explored for transtympanic applications. Ex vivo Franz diffusion cell permeation tests have been used and validated for the permeation of compounds through the skin prior to in vivo studies, but their exploitation for transtympanic delivery is limited. Endolysins, peptidoglycan hydrolases derived from bacterial viruses, are attractive antimicrobials, with promising action against the otitis media pathogen Streptococcus pneumoniae. Liposomal systems, such as transfersomes or PEGylated liposomes, have been shown to enhance drug permeation across the TM. Here, we describe the in vitro characterization of the endolysin-loaded liposomal carriers as well as their ex vivo permeation through TMs. Objectives: The main objective was to develop a delivery system containing an endolysin for a targeted transtympanic treatment of otitis media. To achieve this, it was necessary: i) to encapsulate the endolysin into liposomes for a controlled delivery; and ii) to evaluate the transtympanic permeation ability of the formulations. Materials and Methods: Liposomes composed of 4 mM of L-alpha-lecithin and sodium cholate (5:1) (L:SC) or L-alpha-lecithin and PEG2000 PE (10:1) (L:PEG) loaded with the MSlys endolysin were prepared. The size, polydispersity index (PDI), zeta potential, stability, deformability, encapsulation efficiency, and in vitro MSlys release were determined. The cytotoxicity against fibroblasts and keratinocytes and the efficacy against pneumococcal planktonic and biofilm cells were also evaluated in vitro. Permeation studies were performed in Franz diffusion cells using porcine skin, sheep TMs, and cadaveric human TMs. The amount of MSlys permeated and its antipneumococcal activity were evaluated, and the protein integrity was analyzed by SDS-PAGE. Results and Discussion: The MSlys endolysin was encapsulated into liposomes, with an average efficiency of about 35%. Liposomes with ca. 100 nm and relatively low PDI were produced, with L:PEG formulations being smaller and less polydisperse than L:SC. Both characteristics remained stable for one year at 4 C. Liposomes were shown to be deformable and to provide a controlled release of MSlys over time following a first-order kinetics. No cytotoxicity was observed. Endolysin-loaded liposomes interacted with S. pneumoniae cells, reducing both planktonic and biofilm cultures. The potential of L:PEG over L:SC formulations to transport MSlys was demonstrated in preliminary transdermal assays. The permeation of MSlys across the TMs was enhanced when loaded in PEGylated liposomes. Samples were shown to significantly reduce pneumococcal cells after 2 h of permeation through the human TM. Nonetheless, loss of antipneumococcal activity after 4 h of permeation and protein hydrolysis at 48 and 72 h were observed. Conclusions: This work reports the delivery of an endolysin through an intact TM using liposomes. However, further optimization is needed to expand the overall therapeutic efficacy of this strategy for use in otitis media.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, the Wyss Institute for Biologically Inspired Engineering and from NIH NIDCD K08-DC018575 grant. MDS acknowledges the FCT grants SFRH/BD/128825/2017 and COVID/BD/152363/2022, and SS acknowledges funding by FCT through the individual scientific employment program contract (2020.03171.CEECIND).info:eu-repo/semantics/publishedVersio

Rhamnolipids-based nanostructured lipid carriers: Effect of lipid phase on physicochemical properties and stability

In this work rhamnolipids were evaluated as surfactants for the production of nanostructured lipid carriers (NLCs). NLCs were produced by melt-emulsification using ultra-homogenisation followed by ultrasonication and different ratios of medium-chain-triglycerides and glycerol monostearate (lipid phase) were tested. NLCs presented sizes and polydispersity index values ranged between 97 and 120 nm and 0.200.26, respectively. Transmission electron microscopy observations confirmed the size and the spherical morphology of the NLCs. The thermal analysis and X-ray diffraction showed that the amount of solid lipid (glycerol monostearate) influences the melting, crystallisation and enthalpy of NLCs and their degree of crystallinity. Results showed that NLCs were more stable at 4 °C and the best formulation (1% of water phase, 0.05% of biosurfactant and solid:liquid ratio of 10:90) was stable for 30 days. This work showed the possibility of using rhamnolipids to produce NLCs and represent an important step for the development of lipid-based nanosystems using biosurfactants.Maria A. Azevedo (SFRH/BD/123364/2016) is the recipient of a fellowship from Fundação para a Ciência e Tecnologia (FCT, Portugal). This study was supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01- 0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors would like to thank the H2020 MSCA-RISE project FODIAC—Food for Diabetes and Cognition (reference number 778388) and the Comissão de Coordenação e Desenvolvimento Regional do Norte (CCDR-N) project “Nanotechnology based functional solutions” (No. NORTE01- 0145-FEDER-000019). The authors would like to acknowledge to Oliver Schraidt from INL for his kind assistance using TEM.info:eu-repo/semantics/publishedVersio

Microfluidics for controlled self-assembly of cubosome nanoparticles of tunable size

info:eu-repo/semantics/publishedVersio

In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and the Project FCOMP-01– 0124-FEDER-021053 (PTDC/SAU-BMA/121028/2010). This research was also supported by the Microfluidic Layer-by-layer Assembly of Cationic Liposome—Nucleic Acid Nanoparticles for Gene Delivery project (032520) co-funded by FCT and the ERDF through COMPETE2020. Diana A. Sousa (D.A.S) and Celso J.O. Ferreira (C.J.O.F) acknowledge FCT for the grants PD/BD/139083/2018 and SFRH/BD/149199/2019, respectively.info:eu-repo/semantics/publishedVersio

Magnetic response dependence of ZnO based thin films on Ag doping and processing architecture

Multifunctional and multiresponsive thin films are playing an increasing role in modern technology. This work reports a study on the magnetic properties of ZnO and Ag-doped ZnO semiconducting films prepared with a zigzag-like columnar architecture and their correlation with the processing conditions. The films were grown through Glancing Angle Deposition (GLAD) co-sputtering technique to improve the induced ferromagnetism at room temperature. Structural and morphological characterizations have been performed and correlated with the paramagnetic resonance measurements, which demonstrate the existence of vacancies in both as-cast and annealed films. The magnetic measurements reveal changes in the magnetic order of both ZnO and Ag-doped ZnO films with increasing temperature, showing an evolution from a paramagnetic (at low temperature) to a diamagnetic behavior (at room temperature). Further, the room temperature magnetic properties indicate a ferromagnetic order even for the un-doped ZnO film. The results open new perspectives for the development of multifunctional ZnO semiconductors, the GLAD co-sputtering technique enables the control of the magnetic response, even in the un-doped semiconductor materials.The Brazilian agencies CNPq, CAPES partially supports the research. From Portugal side, this work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UID/FIS/04650/2020 and the junior research contract (A.F.). Financial support from the Basque Government Industry Department under the ELKARTEK. HAZITEK and PIBA programs is also acknowledged

Novel nano-engineering phytantriol-F127-based cubosomes for antibiotic delivery

Nanoparticles exhibited exceptional properties for drug delivery via different routes with excellent pharmacokinetics profiles using minimal dosages with almost no systemic side effects. The delivery of drug molecules for therapeutic applications usually faces problems of solubility and bioavailability that cubosomes can easily overcome. Cubosomes are cubic lipid-based nanoparticles with increased surface area, low viscosity and high heat stability. The preparation of cubosomes and drug encapsulation are still procedures requiring important improvements that have limit their use as drug delivery system. This work we developed a simple experimental procedure able to produce controlled-size cubosomes with different hydrodynamic size dependent of the phytantriol/ethanol (lipid/hydrotrope) ratio. Moreover, cubosomes were easily loaded with ciprofloxacin without structural changes. The results of this study will greatly impact on the applicability of cubosomes as drug delivery system.info:eu-repo/semantics/publishedVersio