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Nanomicellar Formulation of Clotrimazole Improves Its Antitumor Action toward Human Breast Cancer Cells.

Marcondes MC, Fernandes AC, Itabaiana I, de Souza RO, Sola-Penna M, Zancan P - PLoS ONE (2015)

Bottom Line: We found that nCTZ was more efficient than sCTZ at inhibiting glycolytic and other cytosolic and mitochondrial enzymes.This was especially evident on regard to antioxidant potential, which is an important cell defense against drugs that affect cell metabolism.Moreover, this water-soluble formulation of CTZ strengths its potential use as an anticancer medicine.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Oncobiologia Molecular (LabOMol), Departamento de Biotecnologia Farmacêutica (BioTecFar), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.

ABSTRACT

Background: Although demonstrated as a selective anticancer drug, the clinical use of clotrimazole (CTZ) is limited due to its low solubility in hydrophilic fluids. Thus, we prepared a water-soluble nanomicellar formulation of CTZ (nCTZ) and tested on the human breast cancer cell line MCF-7 biology.

Methodology/principal findings: CTZ was nanoencapsulated in tween 80 micelles, which generated nanomicelles of, approximately, 17 nm of diameter. MCF-7 cells were treated with nCTZ and unencapsulated DMSO-solubilized drug (sCTZ) was used for comparison. After treatment, the cells were evaluated in terms of metabolism, proliferation, survival and structure. We found that nCTZ was more efficient than sCTZ at inhibiting glycolytic and other cytosolic and mitochondrial enzymes. Moreover, this increased activity was also observed for lactate production, intracellular ATP content, ROS production and antioxidant potential. As a consequence, nCTZ-treated MCF-7 cells displayed alterations to the plasma membrane, mitochondria and the nucleus. Finally, nCTZ induced both apoptosis and necrosis in MCF-7 cells.

Conclusions/significance: MCF-7 cells are more sensible to nCTZ than to sCTZ. This was especially evident on regard to antioxidant potential, which is an important cell defense against drugs that affect cell metabolism. Moreover, this water-soluble formulation of CTZ strengths its potential use as an anticancer medicine.

No MeSH data available.


Related in: MedlinePlus

Size characterization and initial effects of CTZ-containing nanomicelles.Nanomicelles were prepared as described in Materials and Methods in the presence or absence of CTZ. The size of the nanomicelles was analyzed by dynamic light scattering as described above. Panel A: nanomicelles prepared in the absence of CTZ. Panel B: CTZ-containing nanomicelles. Panel C: MCF-7 cell viability evaluated by the MTT assay in the presence of soluble CTZ (black circles) or a nanomicellar preparation of the drug (white circles). Panel D: MCF-7 cell viability evaluated by LDH activity that leaked from the cells in the presence of soluble CTZ (black circles) or a nanomicellar preparation of the drug (white circles). Plotted points are the means ± standard error of the mean, for at least 4 independent experiments (n = 4).
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pone.0130555.g001: Size characterization and initial effects of CTZ-containing nanomicelles.Nanomicelles were prepared as described in Materials and Methods in the presence or absence of CTZ. The size of the nanomicelles was analyzed by dynamic light scattering as described above. Panel A: nanomicelles prepared in the absence of CTZ. Panel B: CTZ-containing nanomicelles. Panel C: MCF-7 cell viability evaluated by the MTT assay in the presence of soluble CTZ (black circles) or a nanomicellar preparation of the drug (white circles). Panel D: MCF-7 cell viability evaluated by LDH activity that leaked from the cells in the presence of soluble CTZ (black circles) or a nanomicellar preparation of the drug (white circles). Plotted points are the means ± standard error of the mean, for at least 4 independent experiments (n = 4).

Mentions: With the intention of circumventing the major issue hindering the therapeutic use of CTZ, i.e. its low solubility, we incorporated CTZ into a nanoscale water-soluble Tween 80 emulsion, as described in Materials and Methods. The physical characterization of these nanoparticles reveals that they have diameters of 16.9 ± 3.4 nm without CTZ and 17.1 ± 3.9 nm for when CTZ is incorporated (Fig 1A and 1B, respectively). That CTZ incorporation does not significantly affect the nanoparticles’ size is indicative of the stability of the formulation. Indeed, CTZ-containing nanoparticles are stable for at least 24 hours at room temperature (data not shown). To evaluate the efficiency of drug delivery, MCF-7 cells were grown to confluence and incubated for 24 hours in the presence of different concentrations of a DMSO-soluble CTZ (sCTZ) preparation or a nanomicellar CTZ (nCTZ) preparation. After incubation, cell viability was tested with the MTT assay (Fig 1C) and by the leakage of LDH into the culture medium (Fig 1D). Both techniques revealed that nCTZ was able to affect MCF-7 viability more efficiently than sCTZ, based on the concentrations necessary to significantly affect viability (the half-maximal effect was achieved at approximately 25 μM nCTZ and 50 μM sCTZ) and the maximum effects for each (sCTZ promoted approximately half of the maximum effect observed for nCTZ).


Nanomicellar Formulation of Clotrimazole Improves Its Antitumor Action toward Human Breast Cancer Cells.

Marcondes MC, Fernandes AC, Itabaiana I, de Souza RO, Sola-Penna M, Zancan P - PLoS ONE (2015)

Size characterization and initial effects of CTZ-containing nanomicelles.Nanomicelles were prepared as described in Materials and Methods in the presence or absence of CTZ. The size of the nanomicelles was analyzed by dynamic light scattering as described above. Panel A: nanomicelles prepared in the absence of CTZ. Panel B: CTZ-containing nanomicelles. Panel C: MCF-7 cell viability evaluated by the MTT assay in the presence of soluble CTZ (black circles) or a nanomicellar preparation of the drug (white circles). Panel D: MCF-7 cell viability evaluated by LDH activity that leaked from the cells in the presence of soluble CTZ (black circles) or a nanomicellar preparation of the drug (white circles). Plotted points are the means ± standard error of the mean, for at least 4 independent experiments (n = 4).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476588&req=5

pone.0130555.g001: Size characterization and initial effects of CTZ-containing nanomicelles.Nanomicelles were prepared as described in Materials and Methods in the presence or absence of CTZ. The size of the nanomicelles was analyzed by dynamic light scattering as described above. Panel A: nanomicelles prepared in the absence of CTZ. Panel B: CTZ-containing nanomicelles. Panel C: MCF-7 cell viability evaluated by the MTT assay in the presence of soluble CTZ (black circles) or a nanomicellar preparation of the drug (white circles). Panel D: MCF-7 cell viability evaluated by LDH activity that leaked from the cells in the presence of soluble CTZ (black circles) or a nanomicellar preparation of the drug (white circles). Plotted points are the means ± standard error of the mean, for at least 4 independent experiments (n = 4).
Mentions: With the intention of circumventing the major issue hindering the therapeutic use of CTZ, i.e. its low solubility, we incorporated CTZ into a nanoscale water-soluble Tween 80 emulsion, as described in Materials and Methods. The physical characterization of these nanoparticles reveals that they have diameters of 16.9 ± 3.4 nm without CTZ and 17.1 ± 3.9 nm for when CTZ is incorporated (Fig 1A and 1B, respectively). That CTZ incorporation does not significantly affect the nanoparticles’ size is indicative of the stability of the formulation. Indeed, CTZ-containing nanoparticles are stable for at least 24 hours at room temperature (data not shown). To evaluate the efficiency of drug delivery, MCF-7 cells were grown to confluence and incubated for 24 hours in the presence of different concentrations of a DMSO-soluble CTZ (sCTZ) preparation or a nanomicellar CTZ (nCTZ) preparation. After incubation, cell viability was tested with the MTT assay (Fig 1C) and by the leakage of LDH into the culture medium (Fig 1D). Both techniques revealed that nCTZ was able to affect MCF-7 viability more efficiently than sCTZ, based on the concentrations necessary to significantly affect viability (the half-maximal effect was achieved at approximately 25 μM nCTZ and 50 μM sCTZ) and the maximum effects for each (sCTZ promoted approximately half of the maximum effect observed for nCTZ).

Bottom Line: We found that nCTZ was more efficient than sCTZ at inhibiting glycolytic and other cytosolic and mitochondrial enzymes.This was especially evident on regard to antioxidant potential, which is an important cell defense against drugs that affect cell metabolism.Moreover, this water-soluble formulation of CTZ strengths its potential use as an anticancer medicine.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Oncobiologia Molecular (LabOMol), Departamento de Biotecnologia Farmacêutica (BioTecFar), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.

ABSTRACT

Background: Although demonstrated as a selective anticancer drug, the clinical use of clotrimazole (CTZ) is limited due to its low solubility in hydrophilic fluids. Thus, we prepared a water-soluble nanomicellar formulation of CTZ (nCTZ) and tested on the human breast cancer cell line MCF-7 biology.

Methodology/principal findings: CTZ was nanoencapsulated in tween 80 micelles, which generated nanomicelles of, approximately, 17 nm of diameter. MCF-7 cells were treated with nCTZ and unencapsulated DMSO-solubilized drug (sCTZ) was used for comparison. After treatment, the cells were evaluated in terms of metabolism, proliferation, survival and structure. We found that nCTZ was more efficient than sCTZ at inhibiting glycolytic and other cytosolic and mitochondrial enzymes. Moreover, this increased activity was also observed for lactate production, intracellular ATP content, ROS production and antioxidant potential. As a consequence, nCTZ-treated MCF-7 cells displayed alterations to the plasma membrane, mitochondria and the nucleus. Finally, nCTZ induced both apoptosis and necrosis in MCF-7 cells.

Conclusions/significance: MCF-7 cells are more sensible to nCTZ than to sCTZ. This was especially evident on regard to antioxidant potential, which is an important cell defense against drugs that affect cell metabolism. Moreover, this water-soluble formulation of CTZ strengths its potential use as an anticancer medicine.

No MeSH data available.


Related in: MedlinePlus