The Possible "Proton Sponge " Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH.

Polycations such as polyethylenimine (PEI) are used in many novel nonviral vector designs and there are continuous efforts to increase our mechanistic understanding of their interactions with cells. Even so, the mechanism of polyplex escape from the endosomal/lysosomal pathway after internalization is still elusive. The “proton sponge ” hypothesis remains the most generally accepted mechanism, although it is heavily debated. This hypothesis is associated with the large buffering capacity of PEI and other polycations, which has been interpreted to cause an increase in lysosomal pH even though no conclusive proof has been provided. In the present study, we have used a nanoparticle pH sensor that was developed for pH measurements in the endosomal/lysosomal pathway. We have carried out quantitative measurements of lysosomal pH as a function of PEI content and correlate the results to the “proton sponge ” hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the “proton sponge ” effect is the dominant mechanism of polyplex escape

Comparison of gene transfection and cytotoxicity mechanisms of linear poly(amidoamine) and branched poly(ethyleneimine) polyplexes

Purpose: This study aimed to further explore the mechanisms behind the ability of certain linear polyamidoamines (PAAs) to transfect cells with minimal cytotoxicity. Methods: The transfection efficiency of DNA complexed with a PAA of a molecular weight over 10 kDa or 25 kDa branched polyethyleneimine (BPEI) was compared in A549 cells using a luciferase reporter gene assay. The impact of endo/lysosomal escape on transgene expression was investigated by transfecting cells in presence of bafilomycin A1 or chloroquine. Cytotoxicity caused by the vectors was evaluated by measuring cell metabolic activity, lactate dehydrogenase release, formation of reactive oxygen species and changes in mitochondrial membrane potential. Results: The luciferase activity was 3-fold lower after transfection with PAA polyplexes than with BPEI complexes at the optimal polymer to nucleotide ratio (RU:Nt). However, in contrast to BPEI vectors, PAA polyplexes caused negligible cytotoxic effects. The transfection efficiency of PAA polyplexes was significantly reduced in presence of bafilomycin A1 while chloroquine enhanced or decreased transgene expression depending on the RU:Nt. Conclusions: PAA polyplexes displayed a pH-dependent endo/lysosomal escape which was not associated with cytotoxic events, unlike observed with BPEI polyplexes. This is likely due to their greater interactions with biological membranes at acidic than neutral pH

Abstracts from the 20th International Symposium on Signal Transduction at the Blood-Brain Barriers

https://deepblue.lib.umich.edu/bitstream/2027.42/138963/1/12987_2017_Article_71.pd

Duración del tratamiento con etanercept y razones de discontinuación en una cohorte de pacientes con patología reumática

[EN] Objective: To evaluate the duration of etanercept (ETN) treatment and motives for discontinuation in our local cohort of patients with rheumatic pathology and compare them to the group with other biological treatments. Patients and methods: Prospective observational cohort study. Disease diagnosis, start and end date and motive for discontinuation were recorded. Survival estimation was explored using Kaplan-Meier analysis with remaining patients censored at 1-year, 2-years and 5-years follow-up. Results: Ninety-two (45%) out of 205 patients started ETN treatment. Disease diagnoses recorded were: 48% rheumatoid arthritis, 33% ankylosing spondylitis, 11% psoriatic arthritis, 8% others (juvenile idiopathic arthritis, inflammatory bowel disease related spondylitis, SAPHO syndrome). 52% of patients are still on the drug. The motives for discontinuation were: inefficacy (65%), adverse events (33%) and lack of compliance (2%). Two patients discontinued ETN due to prolonged disease control. Adverse events were: infection (4 patients), post-injection skin reaction (3), uveitis (3), neoplasia (2) and others (3). Using a Kaplan–Meier analysis, at 1-year 64% (CI95% 54-74) of patients with ETN treatment had not experienced treatment failure, at 2-years, 59% (48-69) and at 5-years, 43% (30-52). With the rest of biologicals estimated survival was 61% (51-68), 47,5% (40-55) and 23% (10,5-32) respectively. Statistical analysis revealed significant differences (log-rank: P=.024; Breslow: P=.068; Tarone-Ware: P = .040). Conclusions: In our cohort of patients treated with ETN the estimated survival was better than patients treated with other biological drugs at 1-year, 2-years and 5-years. © 2011 Elsevier Espana, S.L. All rights reserved.[ES] Objetivos: Evaluar la supervivencia del tratamiento con etanercept (ETN) y las causas de discontinuación en una cohorte local de pacientes en tratamiento biológico (TB). Comparar con la supervivencia general del resto de TB. Pacientes y métodos: Estudio observacional prospectivo de cohortes. Se han analizado los datos de diagnóstico, fecha de inicio y fin de tratamiento, así como la causa de interrupción de nuestro registro de TB. Mediante el método de Kaplan-Meier se ha estimado la supervivencia de ETN al ano, a los 2 a ˜ nos y a los ˜ 5 anos. ˜ Resultados: De un total de 205 pacientes que recibieron TB, 92 (45%) iniciaron tratamiento con ETN. En el 48% el diagnóstico fue artritis reumatoide, 33% espondilitis anquilosante, 11% artritis psoriásica y 8% otros diagnósticos (artritis idiopática juvenil, espondiloartritis asociada a enfermedad inflamatoria intestinal y síndrome SAPHO). Continúan con ETN 48 pacientes (52%). Las causas de discontinuación fueron: ineficacia (65%), acontecimiento adverso (33%), pérdida de seguimiento (2%). En 2 pacientes el tratamiento se retiró por remisión clínica. Los acontecimientos adversos fueron: infección (4 pacientes), reacción cutánea postinyección (3), uveítis (3), neoplasia (2) y otros (3). La supervivencia estimada de ETN al ano de tratamiento ˜ fue del 64% (IC del 95%, 54-74), a los dos anos del 59% (48-69 ˜ ) y a los 5 anos del 43% (30-52), y la del resto ˜ de TB fue del 61% (51-68), el 47,5% (40-55) y el 23% (10,5-32), respectivamente. Los tests estadísticos revelaron diferencias significativas (log-rank: p = 0,024; Breslow: p = 0,068; Tarone-Ware: p = 0,040). Conclusiones: En nuestra cohorte de pacientes la supervivencia estimada de ETN en el primero, segundo y quinto de ano de tratamiento es superior a la obtenida con el resto de TB, siendo la diferencia significativa ˜ a los 5 anos. ˜ © 2011 Elsevier Espana, S.L. Todos los derechos reservados.Senabre-Gallego, JM.; Rosas, J.; Santos-Soler, G.; Santos Ramirez, C.; Sánchez-Barrioluengo, M.; Salas, E.; Barber, X.... (2011). Duración del tratamiento con etanercept y razones de discontinuación en una cohorte de pacientes con patología reumática. Reumatología Clínica. 7(6):385-388. doi:10.1016/j.reuma.2011.06.0053853887