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1.
Bioengineering (Basel) ; 11(5)2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38790309

ABSTRACT

This research investigates the suitability of printed polyamide 12 (PA12) and its dyed version to support cells in bioengineering applications. For this purpose, human gingival fibroblasts (hGF06) were cultured on PA-12 scaffolds that were 3D-printed by Multi Jet Fusion (MJF). The study examined the direct cultivation of cells on MJF-printed cell culture scaffolds and the effect of leachate of PA-12 printed by MJF on the cultured cells. The article presents research on the surface treatment of PA12 material used in 3D printing and the effect of automatic staining on cell vitality and proliferation in vitro. The study presents a unique device designed exclusively for staining prints made of the biocompatible material PA12 and demonstrates the compatibility of 3D-printed polyamide 12 parts stained in the novel device with a nutrient culture medium and cells. This novel PA12 surface treatment for biomedical purposes does not affect the compatibility with the culture medium, which is essential for cell viability and proliferation. Fluorescence microscopy revealed that mitochondrial fitness and cell survival were not affected by prolonged incubation with clear or dyed PA12 3D-printed parts.

2.
Methods Mol Biol ; 1264: 183-93, 2015.
Article in English | MEDLINE | ID: mdl-25631014

ABSTRACT

Time-resolved fluorescence spectrometry is a highly valuable technological tool to detect and characterize mitochondrial metabolic oxidative changes by means of endogenous fluorescence (Chorvat and Chorvatova, Laser Phys Lett 6: 175-193, 2009). Here, we describe the detection and measurement of endogenous mitochondrial NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence directly in living cultured cells using fluorescence lifetime spectrometry imaging after excitation with 405 nm picosecond (ps) laser. Time-correlated single photon counting (TCSPC) method is employed.


Subject(s)
Mitochondria/metabolism , NADP/metabolism , Oxidation-Reduction , Spectrometry, Fluorescence/methods , Cell Line , Cell Survival , Electron Transport , Humans , Image Processing, Computer-Assisted , Microscopy, Fluorescence/methods
3.
Apoptosis ; 19(12): 1779-92, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25300800

ABSTRACT

In order to explain the contribution of the protein kinase Cα (PKCα) in apoptosis induced by photo-activation of hypericin (Hyp), a small interfering RNA was used for post-transcriptional silencing of pkcα gene expression. We have evaluated the influence of Hyp photo-activation on cell death in non-transfected and transfected (PKCα(-)) human glioma cells (U-87 MG). No significant differences were detected in cell survival between non-transfected and transfected PKCα(-) cells. However, the type of cell death was notably affected by silencing the pkcα gene. Photo-activation of Hyp strongly induced apoptosis in non-transfected cells, but the level of necrotic cells in transfected PKCα(-) cells increased significantly. The differences in cell death after Hyp photo-activation are demonstrated by changes in: (i) reactive oxygen species production, (ii) Bcl-2 phosphorylation on Ser70 (pBcl-2(Ser70)), (iii) cellular distributions of pBcl-2(Ser70) and (iv) cellular distribution of endogenous anti-oxidant glutathione and its co-localization with mitochondria. In summary, we suggest that post-transcriptional silencing of the pkcα gene and the related decrease of PKCα level considerably affects the anti-apoptotic function and the anti-oxidant function of Bcl-2. This implies that PKCα, as Bcl-2 kinase, indirectly protects U-87 MG cells against oxidative stress and subsequent cell death.


Subject(s)
Apoptosis/drug effects , Perylene/analogs & derivatives , Photosensitizing Agents/pharmacology , Protein Kinase C-alpha/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Anthracenes , Cell Line, Tumor , Cell Survival/drug effects , Gene Expression , Humans , Mitochondria/metabolism , Necrosis , Perylene/pharmacology , Phosphorylation , Protein Kinase C-alpha/antagonists & inhibitors , Protein Kinase C-alpha/genetics , Reactive Oxygen Species/metabolism , Serine/metabolism
4.
Photodiagnosis Photodyn Ther ; 11(2): 213-26, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24583280

ABSTRACT

Hypericin photodynamic therapy (HypPDT) has been found to be an efficient inducer of cell death. However, there are indications that HypPDT also activates rescuing pathways. Cell responses to HypPDT are highly dependent on the Hyp intracellular localization and accumulation. We have shown previously that in U87 MG cells Hyp localizes mostly in ER and partially in mitochondria, lysosomes and Golgi, and that HypPDT resulted primarily in apoptosis via the mitochondrial apoptotic pathway. We have also shown that Hyp co-localizes and interacts with anti-apoptotic PKCα in U87 MG cells. To follow up on our previous work, we investigated how HypPDT influences PKCα in U87 MG cells. Here, we show that majority of PKCα present in U87 MG cells is already in a catalytically competent form phosphorylated at Thr638, and it is a likely Bcl2 kinase. The presence of Hyp itself does not affect PKCα distribution. HypPDT acute effect caused PKCα activation and translocation along the plasma membrane and partially in the nuclei. The prolonged effect of HypPDT, 5 and 24h post PDT, results in PKCα located predominantly in cytosol and nuclei. Moreover, we have shown that phosphorylated catalytically competent PKCα is critical for U87 glioma cell viability in response to HypPDT treatment.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Glioma/drug therapy , Glioma/metabolism , Perylene/analogs & derivatives , Photochemotherapy/methods , Protein Kinase C-alpha/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Anthracenes , Cell Line, Tumor , Glioma/pathology , Humans , Perylene/therapeutic use , Phosphorylation/drug effects , Phosphorylation/radiation effects , Photosensitizing Agents/therapeutic use , Treatment Outcome
5.
Int J Pharm ; 436(1-2): 463-71, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-22814227

ABSTRACT

Low-density lipoproteins (LDL), a natural in vivo carrier of cholesterol in the vascular system, play a key role in the delivery of hydrophobic/amphiphilic photosensitizers to tumor cells in photodynamic therapy of cancer. To make this delivery system even more efficient, we have constructed a nano-delivery system by coating of LDL surface by dextran. Fluorescence spectroscopy, confocal fluorescence imaging, stopped-flow experiments and flow-cytometry were used to characterize redistribution of hypericin (Hyp), a natural occurring potent photosensitizer, loaded in LDL/dextran complex to free LDL molecules as well as to monitor cellular uptake of Hyp by U87-MG cells. It is shown that the redistribution process of Hyp between LDL molecules is significantly suppressed by dextran coating of LDL surface. The modification of LDL molecules by dextran does not inhibit their recognition by cellular LDL receptors and U-87 MG cellular uptake of Hyp loaded in LDL/dextran complex appears to be similar to that one observed for Hyp transported by unmodified LDL particles. Thus, it is proposed that dextran modified LDL molecules could be used as a basis for construction of a drug transport system for targeted delivery of hydrophobic/amphiphilic drugs to cancer cells expressing high level of LDL receptors.


Subject(s)
Dextrans/chemistry , Drug Carriers/chemistry , Lipoproteins, LDL/chemistry , Perylene/analogs & derivatives , Radiation-Sensitizing Agents/chemistry , Anthracenes , Cell Line, Tumor , Dextrans/administration & dosage , Drug Carriers/administration & dosage , Humans , Hydrophobic and Hydrophilic Interactions , Lipoproteins, LDL/administration & dosage , Neoplasms/metabolism , Perylene/administration & dosage , Perylene/chemistry , Radiation-Sensitizing Agents/administration & dosage
6.
Photochem Photobiol Sci ; 11(9): 1428-36, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22729350

ABSTRACT

Hypericin (Hyp) is a hydrophobic natural photosensitizer that is considered to be a promising molecule for photodynamic treatment of tumor cells and photo-diagnosis of early epithelial cancers. Its hydrophobicity is the main driving force that governs its redistribution process. Low-density lipoproteins (LDL), a natural in vivo carrier of cholesterol present in the vascular system, have been used for targeted transport of Hyp to U87 glioma cells. For low Hyp-LDL ratios (≤10 : 1), the cellular uptake of Hyp is characterized by endocytosis of the [Hyp-LDL] complex, while Hyp alone can enter cells by passive diffusion. Photo-induced cell death and the mitochondrial membrane potential, observed for glioma cells after various times of incubation with the [Hyp-LDL] complex or Hyp alone, were monitored by flow-cytometry analysis using Annexin-V-FITC propidium iodide and DiOC(6)(3) staining. Differences of the results are discussed in view of the respective dynamic subcellular distributions of the drugs that were obtained by co-localization experiments using confocal fluorescence microscopy. In order to give clear evidence of specific intracellular localization and to identify possible Hyp aggregation in cellular organelles, fluorescence resonance energy transfer (FRET) between selected fluorescent organelle probes and Hyp was also assessed. It is shown, that the observed photo-induced cell deaths can be correlated with the sub-cellular distribution of the active fluorescent monomer form of Hyp in lysosomes (as determined from steady-state fluorescence experiments), but that possible aggregation of Hyp in some organelles, as determined from FRET experiments, should be taken into account for interpretation of the real dynamics of the subcellular redistribution. Results of the present study underline the fact that photo-induced cell death processes are strongly influences by dynamics of Hyp subcellular redistribution processes involving monomer-aggregate equilibrium. Such an observation should be taken in consideration for further optimization of Hyp in vivo PDT applications.


Subject(s)
Apoptosis/drug effects , Organelles/metabolism , Perylene/analogs & derivatives , Photosensitizing Agents/toxicity , Anthracenes , Cell Line, Tumor , Endocytosis , Fluorescence Resonance Energy Transfer , Glioma/metabolism , Glioma/pathology , Humans , Lipoproteins, LDL/chemistry , Lipoproteins, LDL/metabolism , Membrane Potential, Mitochondrial/drug effects , Microscopy, Confocal , Perylene/chemistry , Perylene/toxicity , Photosensitizing Agents/chemistry
7.
Int J Pharm ; 389(1-2): 32-40, 2010 Apr 15.
Article in English | MEDLINE | ID: mdl-20083174

ABSTRACT

The natural photosensitizer hypericin exhibits potent properties for tumor diagnosis and photodynamic therapy. Fluorescent properties of hypericin along with various technical approaches have been used for dynamic studies of its interaction with low-density lipoprotein and U87 glioma cells. Evidences for hypericin release from low-density lipoprotein towards cells plasmatic membrane are addressed. Subsequent subcellular bulk flow redistribution leading to non-specific staining of intracellular membranes compartment were observed within cells. It was shown, that monomers of hypericin are the only redistributive forms. Increasing concentration of hypericin leads to the formation of non-fluorescent aggregates within low-density lipoprotein as well as within the U87 cells, and can preclude its photosensitizing activities. However, the aggregation process can only account for a part of the observed emission decrease. As shown by the excited state lifetime measurements, this fluorescence quenching actually results from a combination of aggregation process and energy transfer from monomers to aggregates. In all experiments, hydrophobic character of hypericin appears as the driving force of its redistribution process.


Subject(s)
Lipoproteins, LDL/metabolism , Perylene/analogs & derivatives , Photosensitizing Agents/chemistry , Anthracenes , Cell Line, Tumor , Cell Membrane/metabolism , Fluorescence , Glioma , Humans , Hydrophobic and Hydrophilic Interactions , Perylene/chemistry , Perylene/pharmacokinetics , Photosensitizing Agents/pharmacokinetics
8.
Photochem Photobiol ; 84(1): 120-7, 2008.
Article in English | MEDLINE | ID: mdl-18173711

ABSTRACT

The dependence of the uptake of hypericin (Hyp) by human glioma U-87 MG cells on the level of expression of low-density lipoprotein (LDL) receptors has been studied in this work. A special role of the LDL receptor-pathway for Hyp delivery to U-87 MG cells in the presence of LDL was revealed by the substantial increase of Hyp uptake in the situation, when the number of LDL receptors on the cell surface was elevated. Moreover, the colocalization experiments showed the lysosomal localization of Hyp following the uptake and that the concentration of Hyp in these organelles was enhanced in the cells with elevated number of LDL receptors when the incubation medium contained LDL. Both these findings suggest that LDL and LDL receptor-pathway play an important role in the delivery and accumulation of Hyp into the cells.


Subject(s)
Cholesterol, LDL/metabolism , Perylene/analogs & derivatives , Receptors, LDL/metabolism , Anthracenes , Cell Line, Tumor , Humans , Lysosomes , Molecular Structure , Perylene/chemistry , Perylene/metabolism
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