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Background: Lenvatinib, a tyrosine kinase inhibitor (TKI) approved for the treatment of progressive and radioactive iodine (RAI)-refractory differentiated thyroid cancer (DTC), is associated with significant adverse effects that can be partially mitigated through the development of novel drug formulations. The utilization of nanoparticles presents a viable option, as it allows for targeted drug delivery, reducing certain side effects and enhancing the overall quality of life for patients. This study aimed to produce and assess, both in vitro and in vivo, the cytotoxicity, biodistribution, and therapeutic efficacy of lenvatinib-loaded PLGA nanoparticles (NPs), both with and without decoration using antibody conjugation (cetuximab), as a novel therapeutic approach for managing aggressive thyroid tumors. Methods: Poly(lactic-co-glycolic acid) nanoparticles (NPs), decorated with or without anti-EGFR, were employed as a lenvatinib delivery system. These NPs were characterized for size distribution, surface morphology, surface charge, and drug encapsulation efficiency. Cytotoxicity was evaluated through MTT assays using two cellular models, one representing normal thyroid cells (Nthy-ori 3-1) and the other representing anaplastic thyroid cells (CAL-62). Additionally, an in vivo xenograft mouse model was established to investigate biodistribution and therapeutic efficacy following intragastric administration. Results: The NPs demonstrated success in terms of particle size, polydispersity index (PDI), zeta potential, morphology, encapsulation efficiency, and cetuximab distribution across the surface. In vitro analysis revealed cytotoxicity in both cellular models with both formulations, but only the decorated NPs achieved an ID50 value in CAL-62 cells. Biodistribution analysis following intragastric administration in xenografted thyroid mice demonstrated good stability in terms of intestinal barrier function and tumor accumulation. Both formulations were generally well tole

Gene therapy and optogenetics are becoming promising tools for treating several nervous system pathologies. Currently, most of these approaches use viral vectors to transport the genetic material inside the cells, but viruses present some potential risks, such as marked immunogenicity, insertional mutagenesis, and limited insert gene size. In this framework, non-viral nanoparticles, such as niosomes, are emerging as possible alternative tools to deliver genetic material, avoiding the aforementioned problems. To determine their suitability as vectors for optogenetic therapies in this work, we tested three different niosome formulations combined with three optogenetic plasmids in rat cortical neurons in vitro. All niosomes tested successfully expressed optogenetic channels, which were dependent on the ratio of niosome to plasmid, with higher concentrations yielding higher expression rates. However, we found changes in the dendritic morphology and electrophysiological properties of transfected cells, especially when we used higher concentrations of niosomes. Our results highlight the potential use of niosomes for optogenetic applications and suggest that special care must be taken to achieve an optimal balance of niosomes and nucleic acids to achieve the therapeutic effects envisioned by these technologies.

Drug adherence is a significant medical issue, often responsible for sub-optimal outcomes during the treatment of chronic diseases such as rheumatoid or psoriatic arthritis. Monoclonal antibodies (which are exclusively given parenterally) have been proven to be an effective treatment in these cases. The use of auto-injectors is an effective strategy to improve drug adherence in parenteral treatments since these pen-like devices offer less discomfort and increased user-friendliness over conventional syringe-based delivery. This study aims to investigate the feasibility of including a monoclonal antibody as a solid formulation inside an auto-injector pen. Specifically, the objective was to evaluate the drug stability after a concentration (to reduce the amount of solvent and space needed) and freeze-drying procedure. A preliminary screening of excipients to improve stability was also performed. The nano-DSC results showed that mannitol improved the stability of the concentrated, freeze-dried antibody in comparison to its counterpart without it. However, a small instability of the CH2 domain was still found for mannitol samples, which will warrant further investigation. The present results serve as a stepping stone towards advancing future drug delivery systems that will ultimately improve the patient experience and associated drug adherence.

Nanodiamonds were combined with niosome, and resulting formulations were named as nanodiasomes, which were evaluated in terms of physicochemical features, cellular internalization, cell viability and transfection efficiency both in in vitro and in in vivo conditions. Such parameters were analyzed at 4 and 25 °C, and at 15 and 30 days after their elaboration. Nanodiasomes showed a particle size of 128 nm that was maintained over time inside the ± 10% of deviation, unless after 30 days of storage at 25 °C. Something similar occurred with the initial zeta potential value, 35.2 mV, being both formulations more stable at 4 °C. The incorporation of nanodiamonds into niosomes resulted in a 4-fold increase of transfection efficiency that was maintained over time at 4 and 25 °C. In vivo studies reported high transgene expression of nanodiasomes after subretinal and intravitreal administration in mice, when injected freshly prepared and after 30 days of storage at 4 °C.

246 p., Gene therapy is based on the delivery of exogenous genetic material into target cells to modulate theexpression of an altered genome in order to treat a specific disease. Lipid nanocarriers, such as niosomesbased on cationic lipids, non-ionic surfactants and ¿helper¿ components, are considered attractivecandidate for non-viral vectors due to their suitable biocompatibility and high versatility. The niosomechemical composition and their elaboration method influence the biophysical properties which have animpact on transfection efficiency and cytotoxicity. Indeed, compounds with specific properties have beenincluded to overcome some disadvantages of niosome formulations, such as chloroquine, which promotesendosomal escape. Gene therapy can be an excellent treatment for many disorders, in particular cysticfibrosis that is an autosomal monogenic recessive disease caused by different mutations in the cysticfibrosis conductance regulator (CFTR) gene. In this doctoral thesis, we focused on the development andin-depth biophysical and biological characterization of non-viral vectors based on cationic niosomes toface cystic fibrosis by gene therapy approach. The data obtained support that the inclusion of thechloroquine molecule in niosome formulations improves the biophysical properties of niosomes withenhanced transfection efficiencies and lower cytotoxicity. In addition, these niosomes are able to increasethe production of functional CFTR protein in cystic fibrosis cells. Furthermore, the development of athree-dimensional scaffold that better mimics the in vivo environment showed utility for evaluating newtreatments and different schedules of administration for cystic fibrosis.

[EN] Nanodiamonds (NDs) are promising materials for gene delivery because of their unique physicochemical and biological features, along with their possibility of combination with other nonviral systems. Our aim was to evaluate the biophysical performance of NDs as helper components of niosomes, named nanodiasomes, to address a potential nonviral gene delivery nanoplatform for therapeutic applications in central nervous system (CNS) diseases. Nanodiasomes, niosomes, and their corresponding complexes, obtained after genetic material addition at different ratios (w/w), were evaluated in terms of physicochemical properties, cellular uptake, intracellular disposition, biocompatibility, and transfection efficiency in HEK-293 cells. Nanodiasomes, niosomes, and complexes fulfilled the physicochemical features for gene therapy applications. Biologically, the incorporation of NDs into niosomes enhanced 75% transfection efficiency (p < 0.001) and biocompatibility (p < 0.05) to values over 90%, accompanied by a higher cellular uptake (p < 0.05). Intracellular trafficking analysis showed higher endocytosis via clathrins (p < 0.05) in nanodiaplexes compared with nioplexes, followed by higher lysosomal colocalization (p < 0.05), that coexisted with endosomal escape properties, whereas endocytosis mediated by caveolae was the most efficient pathway in the case of nanodiaplexes. Moreover, studies in CNS primary cells revealed that nanodiaplexes successfully transfected neuronal and retinal cells. This proof-of-concept study points out that ND integration into niosomes represents an encouraging nonviral nanoplatform strategy for the treatment of CNS diseases by gene therapy.

The adaptation and progress of 3D printing technology toward 3D bioprinting (specifically adapted to biomedical purposes) has opened the door to a world of new opportunities and possibilities in tissue engineering and regenerative medicine. In this regard, 3D bioprinting allows for the production of tailor-made constructs and organs as well as the production of custom implants and medical devices. As it is a growing field of study, currently, the attention is heeded on the optimization and improvement of the mechanical and biological properties of the so-called bioinks/biomaterial inks. One of the strategies proposed is the use of inorganic ingredients (clays, hydroxyapatite, graphene, carbon nanotubes and other silicate nanoparticles). Clays have proven to be useful as rheological and mechanical reinforcement in a wide range of fields, from the building industry to pharmacy. Moreover, they are naturally occurring materials with recognized biocompatibility and bioactivity, revealing them as optimal candidates for this cutting-edge technology. This review deals with the use of clays (both natural and synthetic) for tissue engineering and regenerative medicine through 3D printing and bioprinting. Despite the limited number of studies, it is possible to conclude that clays play a fundamental role in the formulation and optimization of bioinks and biomaterial inks since they are able to improve their rheology and mechanical properties, thus improving printability and construct resistance. Additionally, they have also proven to be exceptionally functional ingredients (enhancing cellular proliferation, adhesion, differentiation and alignment), controlling biodegradation and carrying/releasing actives with tissue regeneration therapeutic activities.

Lipid nanocarriers, such as niosomes, are considered attractive candidates for non-viral gene delivery due to their suitable biocompatibility and high versatility. In this work, we studied the influence of incorporating chloroquine in niosomes biophysical performance, as well as the effect of non-ionic surfactant composition and protocol of incorporation in their biophysical performance. An exhaustive comparative evaluation of three niosome formulations differing in these parameters was performed, which included the analysis of their thermal stability, rheological behavior, mean particle size, dispersity, zeta potential, morphology, membrane packing capacity, affinity to bind DNA, ability to release and protect the genetic material, buffering capacity and ability to escape from artificially synthesized lysosomes. Finally, in vitro biological studies were, also, performed in order to determine the compatibility of the formulations with biological systems, their transfection efficiency and transgene expression. Results revealed that the incorporation of chloroquine in niosome formulations improved their biophysical properties and the transfection efficiency, while the substitution of one of the non-ionic surfactants and the phase of addition resulted in less biophysical variations. Of note, the present work provides several biophysical parameters and characterization strategies that could be used as gold standard for gene therapy nanosystems evaluation.

[EN]The aim was to evaluate relevant biophysic processes related to the physicochemical features and gene transfection mechanism when sphingolipids are incorporated into a cationic niosome formulation for non-viral gene delivery to central nervous system. For that, two formulations named niosphingosomes and niosomes devoid of sphingolipid extracts, as control, were developed by the oil-in water emulsion technique. Both formulations and the corresponding complexes, obtained upon the addition of the reporter EGFP plasmid, were physicochemically and biologically characterized and evaluated. Compared to niosomes, niosphingosomes, and the corresponding complexes decreased particle size and increased superficial charge. Although there were not significant differences in the cellular uptake, cell viability and transfection efficiency increased when human retinal pigment epithelial (ARPE-19) cells were exposed to niosphingoplexes. Endocytosis via caveolae decreased in the case of niosphingoplexes, which showed higher co-localization with lysosomal compartment, and endosomal escape properties. Moreover, niosphingoplexes transfected not only primary central nervous system cells, but also different cells in mouse retina, depending on the administration route, and brain cortex. These preliminary results suggest that niosphingosomes represent a promising non-viral vector formulation purposed for the treatment of both retinal and brain diseases by gene therapy approach.

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

The main objective of three-dimensional (3D) bioprinting technology is the development of 3D constructs combining biomaterials and cells to replicate tissues and organs. Using this technique, different kinds of epitheliums can be produced in order to imitate the in vivo environment with more precision than cell cultures. Such scaffolds are usually characterized prior to evaluate cellular response upon the addition of different drugs and genetic materials. The aim of this study was to make use of 3D bioprinting technology to develop scaffolds that mimic the in vivo lung environment to evaluate the capacity of niosomes (non-viral vectors) to deliver genetic material for the treatment of cystic fibrosis (CF) genetic disorder.

Lipid nanocarriers, such as niosomes, are considered attractive candidates for non-viral gene delivery due to their suitable biocompatibility and high versatility. In this work, we studied the influence of incorporating chloroquine in niosomes biophysical performance, as well as the effect of non-ionic surfactant composition and protocol of incorporation in their biophysical performance. An exhaustive comparative evaluation of three niosome formulations differing in these parameters was performed, which included the analysis of their thermal stability, rheological behavior, mean particle size, dispersity, zeta potential, morphology, membrane packing capacity, affinity to bind DNA, ability to release and protect the genetic material, buffering capacity and ability to escape from artificially synthesized lysosomes. Finally, in vitro biological studies were, also, performed in order to determine the compatibility of the formulations with biological systems, their transfection efficiency and transgene expression. Results revealed that the incorporation of chloroquine in niosome formulations improved their biophysical properties and the transfection efficiency, while the substitution of one of the non-ionic surfactants and the phase of addition resulted in less biophysical variations. Of note, the present work provides several biophysical parameters and characterization strategies that could be used as gold standard for gene therapy nanosystems evaluation.

Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of niosome formulations include a cationic lipid that is responsible for the electrostatic interactions with the negatively charged genetic material, a non-ionic surfactant that enhances the long-term stability of the niosome, and a helper component that can be added to improve its physicochemical properties and biological performance. This review is aimed at providing recent information about niosome-based non-viral vectors for gene delivery purposes. Specially, we will discuss the composition, preparation methods, physicochemical properties, and biological evaluation of niosomes and corresponding nioplexes that result from the addition of the genetic material onto their cationic surface. Next, we will focus on the in situ application of such niosomes to deliver the genetic material into immune-privileged tissues such as the brain cortex and the retina. Finally, as future perspectives, non-invasive administration routes and different targeting strategies will be discussed.

Gene therapy strategies based on non-viral vectors are currently considered as a promising therapeutic option for the treatment of cystic fibrosis (CF), being liposomes the most commonly used gene carriers. Niosomes offer a powerful alternative to liposomes due to their higher stability and lower cytotoxicity, provided by their non-ionic surfactant and helper components. In this work, a three-formulation screening is performed, in terms of physicochemical and biological behavior, in CF patient derived airway epithelial cells. The most efficient niosome formulation reaches 28% of EGFP expressing live cells and follows caveolae-mediated endocytosis. Transfection with therapeutic cystic fibrosis transmembrane conductance regulator (CFTR) gene results in 5-fold increase of CFTR protein expression in transfected versus non-transfected cells, which leads to 1.5-fold increment of the chloride channel functionality. These findings highlight the relevance of niosome-based systems as an encouraging non-viral gene therapy platform with potential therapeutic benefits for CF.

Cationic niosomes have become important non-viral vehicles for transporting a good number of small drug molecules and macromolecules. Growing interest shown by these colloidal nanoparticles in therapy is determined by their structural similarities to liposomes. Cationic niosomes are usually obtained from the self-assembly of non-ionic surfactant molecules. This process can be governed not only by the nature of such surfactants but also by others factors like the presence of additives, formulation preparation and properties of the encapsulated hydrophobic or hydrophilic molecules. This review is aimed at providing recent information for using cationic niosomes for gene delivery purposes with particular emphasis on improving the transportation of antisense oligonucleotides (ASOs), small interference RNAs (siRNAs), aptamers and plasmids (pDNA).

Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of niosome formulations include a cationic lipid that is responsible for the electrostatic interactions with the negatively charged genetic material, a non-ionic surfactant that enhances the long-term stability of the niosome, and a helper component that can be added to improve its physicochemical properties and biological performance. This review is aimed at providing recent information about niosome-based non-viral vectors for gene delivery purposes. Specially, we will discuss the composition, preparation methods, physicochemical properties, and biological evaluation of niosomes and corresponding nioplexes that result from the addition of the genetic material onto their cationic surface. Next, we will focus on the in situ application of such niosomes to deliver the genetic material into immune-privileged tissues such as the brain cortex and the retina. Finally, as future perspectives, non-invasive administration routes and different targeting strategies will be discussed.

Cationic niosomes have become important non-viral vehicles for transporting a good number of small drug molecules and macromolecules. Growing interest shown by these colloidal nanoparticles in therapy is determined by their structural similarities to liposomes. Cationic niosomes are usually obtained from the self-assembly of non-ionic surfactant molecules. This process can be governed not only by the nature of such surfactants but also by others factors like the presence of additives, formulation preparation and properties of the encapsulated hydrophobic or hydrophilic molecules. This review is aimed at providing recent information for using cationic niosomes for gene delivery purposes with particular emphasis on improving the transportation of antisense oligonucleotides (ASOs), small interference RNAs (siRNAs), aptamers and plasmids (pDNA).

Cystic fibrosis (CF) is a genetic disease that hampers the lung function. Despite that the main defective gene has been deeply characterized, some relevant concerns still need to be resolved before considering gene therapy as a realistic medical choice. One of the major issues that need to be strongly considered in order to succeed in the search for an effective gene therapy approach for CF is the design of the appropriate genetic material to be delivered. Other relevant factors to take into consideration include the design of safe and effective gene delivery systems, the biological barriers that need to be overcome in order to reach the nucleus of the target cells, and the problems related to the design of a drug formulation suitable for lung delivery purposes. Furthermore, some problems related to the commercialization of gene therapy products also need to be resolved. In this chapter, we discuss the up-to-date strategies to overcome such hurdles in order for gene therapy to become a routine treatment modality for CF.