For the validation of the coupling effectiveness, the obtained supernatants were collected and the free MTX was quantitatively measured by ultraviolet/visible (UV/Vis) spectrometry using an Ultrospec 4300 pro UV/Visible Spectrophotometer (Amersham Pharmacia Biotech, Freiburg, Germany). the uptake and efflux of MTX and MNP. Our results exposed a very heterogeneous and cell line-dependent E-64 response to an exposure with MTX-coupled MNP (MTXCMNP), which was almost comparable to the effectiveness of free MTX in the same cell collection. Moreover, a cell line-specific and preferential uptake of MTXCMNP compared with MNP only was found (probably by receptor-mediated endocytosis), agreeing with the observed cytotoxic effects. Opposed to this, the manifestation pattern of several cell membrane transport proteins mentioned for MTX uptake and efflux was only by inclination in agreement with the cellular toxicity of MTXCMNP in different cell lines. Higher cytotoxic effects were achieved by exposing cells to a combination of MTXCMNP and hyperthermal treatment, compared with MTX or thermo-therapy only. However, the heterogeneity in the response of the tumor cell lines to MTX could not be completely abolished C actually after its combination with MNP and/or hyperthermia C and the application of higher thermal dosages might be necessary. Keywords: magnetic nanoparticles, SPION, in vitro, methotrexate, hyperthermia, breast cancer, bladder malignancy Intro The heterogeneity of tumors dramatically impacts a individuals survival due to a selective response of in a different way dedifferentiated cell populations to the respective tumor treatment.1 Based on this circumstance, the limited efficacy of a single treatment, for example, a single chemotherapeutic drug, is not surprising. For this reason, several chemotherapeutic drugs are usually combined in the clinics in order to target multiple cellular signaling pathways and increase the antitumor effect.2 Nevertheless, their dose in malignancy treatment is restricted due to severe side effects affecting the whole body, as they were mostly applied intravenously and don’t exert their effects solely in the tumor E-64 region. As a consequence, drug-based treatments were often applied in several cycles and used in combination with other treatments like radiation. In spite of several advantages to increase therapeutic efficacy, the problems related to the event of side effects still remain. To conquer these drawbacks, a combination of localized antitumor therapies is definitely preferential. In this regard, E-64 magnetic nanoparticles (MNP) operating as drug service providers after being coupled to (eg, chemotherapeutic) medicines can provide a handy alternate. In particular, systemically applied MNP can be specifically enriched in the tumor region by magnetic causes (magnetic focusing on). Hereto, MNP will be able to deposit their cargo (eg, a coupled chemotherapeutic drug) at the prospective site whereby unwanted side effects can be reduced.3C7 Moreover, MNP can be heated in an alternating magnetic field, allowing a localized sensitization or destruction of tumor cells or tumor cells by hyperthermal or E-64 even thermoablative temperatures.8C11 For magnetic heating purposes, iron oxide MNP having Rabbit Polyclonal to RAB41 a clustered magnetite or maghemite core and an appropriate covering (polyethylene glycol [PEG], dextran, dimercaptosuccinic acid [DMSA], etc) have been shown to show good heating capabilities and biocompatibility.10,12C16 One chemotherapeutic drug that can effectively be coupled to MNP is methotrexate (MTX). By this approach, combinatory treatments consisting of MTX-coupled MNP (MTXCMNP) and magnetic hyperthermia have the capability of interfering with multiple phases of the cell cycle, as MTX is known to act, for example, at the G1/S transition (eg, probably by restoring p53 pathways), whereas hyperthermia treatments are reported to act mostly in later phases like S or M phase.17C21 MTX is a structural analog of folate (antifolate) that E-64 inhibits key enzymes of the purine and pyrimidine synthesis by targeting dihydrofolate reductase and thymidylate synthetase. The inhibition of specific steps of the folate metabolism leads to a depletion of.