RESUMO
Extracellular vesicles (EVs), nanovesicles released by cells to effectively exchange biological information, are gaining interest as drug delivery system. Yet, analogously to liposomes, they show short blood circulation times and accumulation in the liver and the spleen. For tissue specific delivery, EV surfaces will thus have to be functionalized. We present a novel platform for flexible modification of EVs with target-specific ligands based on the avidin-biotin system. Genetic engineering of donor cells with a glycosylphosphatidylinositol-anchored avidin (GPI-Av) construct allows the isolation of EVs displaying avidin on their surface, functionalized with any biotinylated ligand. For proof of concept, GPI-Av EVs were modified with i) a biotinylated antibody or ii) de novo designed and synthesized biotinylated ligands binding carbonic anhydrase IX (CAIX), a membrane associated enzyme overexpressed in cancer. Functionalized EVs showed specific binding and uptake by CAIX-expressing cells, demonstrating the power of the system to prepare EVs for cell-specific drug delivery.
Assuntos
Vesículas Extracelulares , Diagnóstico por ImagemRESUMO
The crude methanolic extract obtained from Verbascum nigrum aerial parts (VNE) and its six fractions (VNF1-VNF6) were initially screened regarding their effects on the viability of immortalized HaCaT keratinocytes and A431 epidermoid carcinoma cells (MTT assay, 24â h). None of the tested samples affected the viability of HaCaT cells in a concentration range of 25-150â µg/mL. VNE and VNF4 exhibited significant cytotoxic effects in A431 cells, with IC50 values of 81.92 and 12.27â µg/mL, respectively; the selectivity index was higher than 10 for VNF4. The untargeted LC/HR-MS/MS metabolite profiling led to the tentative annotation of a total number of 23 compounds. Of these, VNE comprised mainly iridoid glycosides (harpagoside, laterioside, acylated aucubin derivatives), whereas VNF4 showed a high abundance of triterpene saponin glycosides (ilwensisaponins A and C, songarosaponins A and B), constituents known for their selective cytotoxic potential.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Verbascum/química , Linhagem Celular Tumoral , HumanosRESUMO
Novel photoactive (metallo)porphyrins were synthesised and characterised. When irradiated with light at a wavelength greater than 600â nm, these porphyrins act as photosensitisers and show high cytotoxicity towards two different human cancer cell lines with IC50 values down to 0.4â µM. A paramagnetic copper(II) porphyrin is the first photosensitiser to display excellent phototoxicity, explained by the electron paramagnetic resonance (EPR) spin trapping of hydroxy radicals and experimentally confirmed by the discovery of elevated levels of reactive oxygen species (ROS) inside A2780 cells after irradiation with red light. This finding indicates that paramagnetic compounds should be considered for photodynamic therapy (PDT). Furthermore, an additive effect of cisplatin and a zinc porphyrin, both at subtherapeutic concentrations of 0.22â µM, was observed.
Assuntos
Antineoplásicos/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/química , Cisplatino/uso terapêutico , Cisplatino/toxicidade , Complexos de Coordenação/química , Complexos de Coordenação/uso terapêutico , Complexos de Coordenação/toxicidade , Cobre/química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Luz , Neoplasias/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/toxicidade , Porfirinas/toxicidade , Espécies Reativas de Oxigênio/metabolismoRESUMO
Epileptic seizures are among the presenting clinical signs of malignant glioma patients, frequently necessitating treatment with antiepileptic drugs (AEDs). The efficacy of 5-aminolevulinic acid (5-ALA)-based intraoperative fluorescence-guided surgery and photodynamic therapy (PDT) in glioblastoma multiforme (GBM) patients depends on the specific accumulation and total amount of intracellularly synthesized protoporphyrin IX (PpIX) in tumour cells. In this study, we investigated the effect of the AEDs phenytoin (PHY) and levetiracetam (LEVE) on 5-ALA-induced PpIX accumulation in two glioma cell lines (U373 MG and U-87 MG) and primary GBM cells isolated from a human biopsy. After treatment with PHY and LEVE for three days cells were incubated with 1 mM: 5-ALA for 4 h and PpIX accumulation was determined by fluorescence measurement. We observed a decrease in PpIX synthesis of up to 55 ± 12 % in primary GBM cells after incubation with phenytoin. This reduction was dose-dependent for all tested cell lines and primary GBM cells. LEVE on the other hand did not alter PpIX concentration in GBM cells. PDT was performed in vitro by irradiating the GBM cells with light doses from 0.5 to 10 J cm(-2) at 627 nm after AED and 5-ALA treatment. Although less PpIX accumulated in PHY-treated cells, efficacy of PDT was not affected. We assume that damage to the mitochondrial membrane by PHY inhibits PpIX synthesis in vitro, because we showed mitochondrial dysfunction as a result of reduced mitochondrial membrane potential in PHY-treated cells. No change in glutathione status was observed. To evaluate further the effect of PHY on PpIX fluorescence, and to establish its significance in clinical practice, animal and clinical studies are required, because the results presented here imply PHY may reduce intracellular accumulation of PpIX in patients with high-grade gliomas.
Assuntos
Ácido Aminolevulínico/farmacologia , Anticonvulsivantes/farmacologia , Glioma/tratamento farmacológico , Glioma/metabolismo , Fenitoína/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/metabolismo , Fluorescência , Glutationa/metabolismo , Humanos , Levetiracetam , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fotoquimioterapia , Piracetam/análogos & derivados , Piracetam/farmacologia , Células Tumorais CultivadasRESUMO
OBJECTIVES: The partial pressure of oxygen (pO2) in brain tumors ranges from 5 to 15%. Nevertheless, the majority of in vitro experiments with glioblastoma multiforme (GBM) cell lines are carried out under an atmospheric pO2 of 19 to 21%. Recently, 5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), has been introduced to neurosurgery to allow for photodynamic diagnosis and photodynamic therapy (PDT) in high-grade gliomas. Here, we investigate whether low pO2 affects GBM cell physiology, PpIX accumulation, or PDT efficacy. METHODS: GBM cell lines (U-87 MG and U-251 MG) were cultured under atmospheric (pO2 â=â 19%) and physiological (pO2 â=â 9%) oxygen concentrations. PpIX accumulation and localization were investigated, and cell survival and cell death were observed following in vitro PDT. RESULTS: A physiological pO2 of 9% stimulated GBM cell migration, increased hypoxia-inducible factor (HIF)-1 alpha levels, and elevated resistance to camptothecin in U-87 MG cells compared to cultivation at a pO2 of 19%. This oxygen reduction did not alter 5-ALA-induced intracellular PpIX accumulation. However, physiological pO2 changed the responsiveness of U-87 MG but not of U-251 MG cells to in vitro PDT. Around 20% more irradiation light was required to kill U-87 MG cells at physiological pO2, resulting in reduced lactate dehydrogenase (LDH) release (one- to two-fold) and inhibition of caspase 3 activation. DISCUSSION: Reduction of oxygen concentration from atmospheric to a more physiological level can influence the malignant behavior and survival of GBM cell lines after in vitro PDT. Therefore, precise oxygen concentration control should be considered when designing and performing experiments with GBM cells.
Assuntos
Técnicas de Cultura de Células/métodos , Glioblastoma/metabolismo , Glioblastoma/terapia , Oxigênio/metabolismo , Fotoquimioterapia , Idoso , Ácido Aminolevulínico/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Caspase 3/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Proteínas da Matriz Extracelular/farmacologia , Feminino , Glioblastoma/fisiopatologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Protoporfirinas/metabolismoRESUMO
5-Aminolevulinic acid (5-ALA) is a natural precursor of protoporphyrin IX (PpIX), which can be used as a photosensitizer in photodynamic therapy (PDT). Accumulation of PpIX in benign meningioma cells has been observed previously, its exploitation for PDT, however, was discouraged by inconsistent results. To evaluate PDT for benign meningiomas, we investigated PpIX synthesis in two human meningioma cell lines (HBL-52 and BEN-MEN-1), their respective extracellular loss of PpIX and corresponding ferrochelatase (FECH) activity as well as their susceptibility to PDT. We demonstrated PpIX production after 5-ALA administration and minor loss to the extracellular space in both cell lines. However, significantly more (up to five times) PpIX was accumulated in BEN-MEN-1 as compared with HBL-52 cells. FECH activity was 2.7-fold higher in HBL-52 compared with BEN-MEN-1 cells and accordingly higher FECH levels could be shown in HBL-52 cells by Western blot analysis. BEN-MEN-1 cells were much more sensitive to PDT and cells could be almost completely killed by irradiation doses of 2 J cm(-2) , whereas HBL-52 showed only an insufficient response at this irradiation dose. We conclude that differences in intracellular PpIX concentrations between HBL-52 and BEN-MEN-1 benign meningioma cells were mainly due to differences in FECH activity and that these differences correspond to their susceptibility to 5-ALA-induced PDT.