GBM is often not feasible due to its location in significant areas of
the brain. GBM, characterized by high degree of invasiveness,
readily infiltrate surrounding tissues, leading to later disease progression or recurrence [3]. It is therefore important to limit the
migration potential of GBM cells. To initiate the migration process
glioma cells degrade the surrounding extracellular matrix (ECM)
into a migration favorable microenvironment [4].
Tenascin-C (TN-C), one of the ECM proteins responsible for
adhesion and invasiveness of cancer cells, is highly overexpressed in
GBM compared to healthy tissues, what makes it a great therapeutic target [5]. The identification of new molecular biomarkers in
GBM as well as new drug delivery strategies are now a major
therapeutic challenge. One of the most promising cancer treatment
methods is gene therapy based on RNA interference (RNAi), referring to post-transcriptional gene silencing mediated by either degradation or translation inhibition of target RNA.
RNAi is triggered by the introduction of double-stranded RNA
(dsRNA) into a cell [6]. In experimental approach, dsRNA with
sequence homologous to TN-C mRNA was used to reduce TN-C
expression [7]. Use of this RNA agent, named ATN-RNA, administrated locally into the tumor’s cavity during standard neurosurgical procedure, results in increased survival and better quality of life
of patients [8]. To overcome the lack of an effective delivery
method for dsRNA and the instability of the nucleic acids during
the delivery, we successfully tested the magnetic nanoparticles
modified with polyethyleneimine (MNP@PEI) as a therapeutics’
carrier [9].
Here, we present a protocol for application of MNP@PEI as a
carrier for ATN-RNA to GBM cells. We demonstrate the protocols
for magnetic nanoparticles synthesis, for assessment of cytotoxicity
of obtained materials, preparation of nanoparticles-dsRNA complexes and their application in delivery of dsRNA to GBM cells. We
also describe a method for assessing the gene silencing level and an
approach for detection of the cell migration impairment.
2 Materials
2.1 Magnetic
Nanoparticles
Synthesis
1. Working solution: 0.5 g of 25-kDa branched polyethyleneimine (PEI-25 Br ), 250 μL of Capstone FS-65 (Du Pont) fluorosurfactant, 2.5 mL of NH 4 OH, 10 mL of water.
70
Małgorzata Grabowska et al.
the brain. GBM, characterized by high degree of invasiveness,
readily infiltrate surrounding tissues, leading to later disease progression or recurrence [3]. It is therefore important to limit the
migration potential of GBM cells. To initiate the migration process
glioma cells degrade the surrounding extracellular matrix (ECM)
into a migration favorable microenvironment [4].
Tenascin-C (TN-C), one of the ECM proteins responsible for
adhesion and invasiveness of cancer cells, is highly overexpressed in
GBM compared to healthy tissues, what makes it a great therapeutic target [5]. The identification of new molecular biomarkers in
GBM as well as new drug delivery strategies are now a major
therapeutic challenge. One of the most promising cancer treatment
methods is gene therapy based on RNA interference (RNAi), referring to post-transcriptional gene silencing mediated by either degradation or translation inhibition of target RNA.
RNAi is triggered by the introduction of double-stranded RNA
(dsRNA) into a cell [6]. In experimental approach, dsRNA with
sequence homologous to TN-C mRNA was used to reduce TN-C
expression [7]. Use of this RNA agent, named ATN-RNA, administrated locally into the tumor’s cavity during standard neurosurgical procedure, results in increased survival and better quality of life
of patients [8]. To overcome the lack of an effective delivery
method for dsRNA and the instability of the nucleic acids during
the delivery, we successfully tested the magnetic nanoparticles
modified with polyethyleneimine (MNP@PEI) as a therapeutics’
carrier [9].
Here, we present a protocol for application of MNP@PEI as a
carrier for ATN-RNA to GBM cells. We demonstrate the protocols
for magnetic nanoparticles synthesis, for assessment of cytotoxicity
of obtained materials, preparation of nanoparticles-dsRNA complexes and their application in delivery of dsRNA to GBM cells. We
also describe a method for assessing the gene silencing level and an
approach for detection of the cell migration impairment.
2 Materials
2.1 Magnetic
Nanoparticles
Synthesis
1. Working solution: 0.5 g of 25-kDa branched polyethyleneimine (PEI-25 Br ), 250 μL of Capstone FS-65 (Du Pont) fluorosurfactant, 2.5 mL of NH 4 OH, 10 mL of water.
70
Małgorzata Grabowska et al.