iPSC-Derived Intestinal Organoids from Cystic Fibrosis Patients Acquire CFTR Activity upon TALEN-Mediated Repair of the p.F508del Mutation.

Cystic fibrosis (CF) is the main genetic cause of death among the Caucasian population. The disease is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth (meconium ileus), later extending to other organs, generally affecting the respiratory tract. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (cAMP)-dependent, phosphorylation-regulated chloride channel required for transport of chloride and other ions through cell membranes. There are more than 2,000 mutations described in the CFTR gene, but one of them, phenylalanine residue at amino acid position 508 (p.F508del), a recessive allele, is responsible for the vast majority of CF cases worldwide. Here, we present the results of the application of genome-editing techniques to the restoration of CFTR activity in p.F508del patient-derived induced pluripotent stem cells (iPSCs). Gene-edited iPSCs were subsequently used to produce intestinal organoids on which the physiological activity of the restored gene was tested in forskolin-induced swelling tests. The seamless restoration of the p.F508del mutation resulted in normal expression of the mature CFTR glycoprotein, full recovery of CFTR activity, and a normal response of the repaired organoids to treatment with two approved CF therapies: VX-770 and VX-809.

Safety and effectiveness of sodium colistimethate-loaded nanostructured lipid carriers (SCM-NLC) against P. aeruginosa: in vitro and in vivo studies following pulmonary and intramuscular administration.

The usefulness of nanotechnology to increase the bioavailability of drugs and decrease their toxicity may be a tool to deal with multiresistant P. aeruginosa (Mr-Pa) respiratory infections. We describe the preparation and the in vivo efficacy and safety of sodium colistimethate-loaded nanostructured lipid carriers (SCM-NLC) by the pulmonary and intramuscular routes. Nanoparticles showed 1-2 mg/L minimum inhibitory concentration against eight extensively drug-resistant P. aeruginosa strains. In vivo, SCM-NLC displayed significantly lower CFU/g lung than the saline and similar to that of the free SCM, even the dose in SCM-NLC group was lower than free SCM. There was no tissue damage related to the treatments. Biodistribution assessments showed a mild systemic absorption after nebulization and a notorious absorption after IM route. Altogether, it could be concluded that SCM-NLC were effective against P. aeruginosa in vivo, not toxic and distribute efficiently to the lung and liver after pulmonary or intramuscular administrations.

Benznidazole Nanoformulates: A Chance to Improve Therapeutics for Chagas Disease.

This article describes the characterization of various encapsulated formulations of benznidazole, the current first-line drug for the treatment of Chagas disease. Given the adverse effects of benznidazole, safer formulations of this drug have a great interest. In fact, treatment of Chagas disease with benznidazole has to be discontinued in as much as 20% of cases due to side effects. Furthermore, modification of delivery and formulations could have potential effects on the emergence of drug resistance. The trypanocidal activity of new nanostructured formulations of benznidazole to eliminate Trypanosoma cruzi was studied in vitro as well as their toxicity in two cultured mammalian cell lines (HepG2 and Fibroblasts). Nanoparticles tested included nanostructured lipid carriers, solid lipid nanoparticles, liposomes, quatsomes, and cyclodextrins. The in vitro cytotoxicity of cyclodextrins-benznidazole complexes was significantly lower than that of free benznidazole, whereas their trypanocidal activity was not hampered. These results suggest that nanostructured particles may offer improved therapeutics for Chagas disease.

Pulmonary delivery of tobramycin-loaded nanostructured lipid carriers for Pseudomonas aeruginosa infections associated with cystic fibrosis.

Among the pathogens that affect cystic fibrosis (CF) patients, Pseudomonas aeruginosa is the most prevalent. As a way to fight against this infection, nanotechnology has emerged over the last decades as a promising alternative to overcome resistance to antibiotics in infectious diseases. The goal of this work was to elaborate and characterize lipid nanoparticles for pulmonary delivery of tobramycin. Tobramycin-loaded nanostructured lipid carriers (Tb-NLCs) were prepared by hot melt homogenization technique. In addition, nanoparticles labeled with infrared dye (IR-NLCs) were used to investigate their in vivo performance after pulmonary administration. Tb-NLCs displayed a mean diameter size around 250 nm, high drug encapsulation (93%) and sustained release profile. Tb-NLCs showed to be active against clinically isolated P. aeruginosa. Moreover, Tb-NLCs did not decrease cell viability and were able to overcome an artificial mucus barrier in the presence of mucolytics agents. During the in vivo assay, IR-NLCs were administered to several mice by the intratracheal route using a Penn Century device. Next, the biodistribution of the nanoparticles was analyzed at different time points showing a wide nanosystem distribution in the lungs. Altogether, tobramycin-loaded NLCs seem to us an encouraging alternative to the currently available CF therapies.

Sodium colistimethate loaded lipid nanocarriers for the treatment of Pseudomonas aeruginosa infections associated with cystic fibrosis.

Lung impairment is the most life-threatening factor for cystic fibrosis patients. Indeed, Pseudomonas aeruginosa is the main pathogen in the pulmonary infection of these patients. In this work, we developed sodium colistimethate loaded lipid nanoparticles, namely, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), as a strategy to enhance the antimicrobial therapy against P. aeruginosa in cystic fibrosis patients. The nanoparticles obtained displayed a 200-400 nm size, high drug entrapment (79-94%) and a sustained drug release profile. Moreover, both SLN and NLC presented antimicrobial activity against clinically isolated P. aeruginosa. The integrity of the nanoparticles was not affected by nebulization through a mesh vibrating nebulizer. Moreover, lipid nanoparticles appeared to be less toxic than free sodium colistimethate in cell culture. Finally, an in vivo distribution experiment showed that nanoparticles spread homogenously through the lung and there was no migration of lipid nanoparticles to other organs, such as liver, spleen or kidneys.

Cellulose acetate phthalate microparticles containing Vibrio cholerae: steps toward an oral cholera vaccine.

Oral cholera vaccine (OCV) has been recommended in some endemic areas and epidemic situations since 1999. Although safe and effective vaccines are currently on the market, the burden of transport and storage remains an issue. Herein, we report an approach to develop an alternative OCV in the form of a gastro-resistant powder. Heat-killed Vibrio cholerae (VC) was encapsulated with a spray-drying technique at different temperatures. Cellulose acetate phthalate (Aquacoat® CPD) was chosen as the core polymer and the addition of alginate was studied. The microparticles (MPs) produced were characterized by surface morphology, particle size, drug loading, antigenicity and gastro resistance. The MPs obtained were 6 µm in size and had appropriate drug content, ranging from 8.16 to 8.64%. Furthermore, antigenicity was maintained, never dropping below 85%, and enteric properties were achieved for all the formulations. Next, an in vivo study was carried out with Aquacoat® CPD MP prepared at 80 °C with and without alginate. Two different doses were assayed, 30 and 60 mg, and compared to the VC suspension. The evoked immune responses showed that alginate containing MPs, especially at the 30 mg dose, displayed values that were very similar to those of VC. In conclusion, spray-dried alginate VC MPs seem to be a promising step toward a powder-form cholera vaccination.

Repeated dose toxicity study of Vibrio cholerae-loaded gastro-resistant microparticles.

CONTEXT: Microencapsulation of antigens has been extensively studied over the last decades aiming at improving the immunogenicity of vaccine candidates. OBJECTIVE: Addressing microparticles (MPs) toxicity in rats. MATERIAL AND METHODS: Spray-dried Eudragit® L 30 D-55 MPs and Eudragit® L 30 D-55 alginate MPs were elaborated and characterized. MPs obtained were administered to rats, three groups were defined: G1, control group; G2, administered with Vibrio cholerae (VC)-loaded MPs; G3, receiving VC-loaded alginate MPs. Animals received three vaccine doses. Body weight, food and water intake were controlled during the study. Haematological parameters, vibriocidal titres, organ weight and histology in necropsy were also analyzed. RESULTS: All animals grew healthy. Body weight gain, food and water intake and haematological parameters remained within physiological values, showing no treatment-related differences. Moreover, organ weight changes were not detected and animals developed protective vibriocidal titres. CONCLUSION: VC-loaded MPs and VC-loaded alginate MPs have proved to be safe and effective in the assessed conditions.

Biomedical applications of immobilized enzymes: an update.

Immobilized enzymes have been widely studied during the last few decades. Biocatalyst systems may work as biosensors or may be used for the treatment of different diseases. This chapter presents different attempts to immobilize enzymes in the biomedical field, particularly the use of prolidase and superoxide dismutase as two examples of this approach. Although this chapter focuses on liposomes and nanoparticles for the entrapment of these enzymes, the methods detailed here could be adapted for the immobilization of other enzymes with therapeutic purposes.

A new oral vaccine candidate based on the microencapsulation by spray-drying of inactivated Vibrio cholerae.

The aim of this work was to evaluate the microencapsulation by spray-drying of inactivated Vibrio cholerae, using methacrylic copolymers Eudragit® L30D-55 and FS30D. The microparticles obtained presented a particle size around 3.0 µm. The preparation temperature affected the morphology and the antigenicity of microparticles, but it did not affect the V. cholerae content. In vitro release studies showed that in acid medium less than 5% of bacteria was released, and in neutral medium, Eudragit® L30D-55 microparticles released 86% after 24 h, whereas FS30D released less than 30%. Rats inoculated with microparticles exhibited vibriocidal antibody titres. Microencapsulation by spray-drying of inactivated V. cholerae could be proposed as a method to obtain an oral vaccine which provides controlled release of the bacteria.