Surface modified magnetic nanoparticles for immuno-gene therapy of murine mammary adenocarcinoma

Kolektor Group, Nanotesla Institute, Ljubljana, Slovenia.
Biomaterials (Impact Factor: 8.56). 03/2012; 33(17):4379-91. DOI: 10.1016/j.biomaterials.2012.02.061
Source: PubMed


Cancer immuno-gene therapy is an introduction of nucleic acids encoding immunostimulatory proteins, such as cytokine interleukin 12 (IL-12), into somatic cells to stimulate an immune response against a tumor. Various methods can be used for the introduction of nucleic acids into cells; magnetofection involves binding of nucleic acids to magnetic nanoparticles with subsequent exposure to an external magnetic field. Here we show that surface modified superparamagnetic iron oxide nanoparticles (SPIONs) with a combination of polyacrylic acid (PAA) and polyethylenimine (PEI) (SPIONs-PAA-PEI) proved to be safe and effective for magnetofection of cells and tumors in mice. Magnetofection of cells with plasmid DNA encoding reporter gene using SPIONs-PAA-PEI was superior in transfection efficiency to commercially available SPIONs. Magnetofection of murine mammary adenocarcinoma with plasmid DNA encoding IL-12 using SPIONs-PAA-PEI resulted in significant antitumor effect and could be further refined for cancer immuno-gene therapy.

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    • "Then, iron oxide nanoparticles were covalently modified with fluorescein isothiocyanate (FI) and HA via PEI-mediated conjugation chemistry. Prijic et al. [19] have modified the surface of SPIONs with a combination of polyacrylic acid (PAA) and polyethyleneimine (PEI) that proved to be safe and effective for magnetofection of cells and tumours. SPIONs were synthesized by alkaline co-precipitation of ferrous and ferric sulphates in an aqueous solution according to the Massart method. "
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    ABSTRACT: Homogeneous hybrids in which iron oxide nanoparticles are entrapped within polymer structure are of interest for their potential applications in biomedical field, such as diagnostic, therapeutic and theranostic purposes. For this reason, hybrid nanomaterials based on branched polyethyleneimine (PEI) and iron oxide with different ratios were synthesized in a single step by hydrothermal procedure at high pressure and low temperature. Iron oxide is formed in the presence of branched PEI and the interaction between them takes place in the reaction medium. The influence of synthesis parameters on the hybrid formation, as well as chemical and structural properties was studied by means of FTIR, DSC-TG, HRTEM, electron paramagnetic resonance (EPR), magnetic measurements (SQUID) and 57Fe Mössbauer analyses. It has been shown that synthesis parameters influence thermal stability and morphology of the hybrids. FeO(OH) crystallites of 2–5 nm are formed. Iron oxyhydroxide nanoparticles strongly entrapped in PEI structure are obtained. The low and distributed values of the specific spontaneous magnetisation in samples prepared under the same pressure conditions support the presence of very fine FeO(OH) nanoparticles, which formation and magnetic properties are depending on the mass ratio between iron oxide and PEI.
    Materials Chemistry and Physics 05/2015; 161. DOI:10.1016/j.matchemphys.2015.05.018 · 2.26 Impact Factor
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    • "Both studies also showed that treatment of cells with chemical inhibitors of endocytosis does not interfere with the formation DNA–membrane interaction areas. Our results on B16F1 murine melanoma cells, which is a cell line that is easily transfected, especially with GET [50] [51], showed that the treatment of cells with MβCD, a general inhibitor of endocytosis which acts by removing cholesterol from the cell membrane [28] [41], significantly reduced the GET efficacy and at the highest tested concentration (7.5 mM) almost prevented it (Fig. 2A). This was demonstrated by the decrease of the fraction of transfected cells as well as by the decrease of their mean fluorescence intensity (Fig. 2A, B). "
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    ABSTRACT: Application of electric pulses (electroporation/electropermeabilization) is an effective method for gene transfer (i.e. gene electrotransfer (GET)) in vitro and in vivo. Currently, the mechanisms by which the DNA enters the cell are not yet fully understood. Experimental evidence is building up that endocytosis is the main mechanism by which the DNA, which is later expressed, enters the cell. Therefore the aim of our study was to elucidate whether inhibitors of endocytosis, methyl-β-cyclodextrin (MβCD), Concanavalin A (ConA) and Dynasore, can impair the transfection efficacy of GET in vitro in B16F1 murine melanoma and in vivo in m. tibialis cranialis in mice. We show that MβCD - general inhibitor of endocytosis - can almost prevent GET of EGFP-N1 plasmid in vitro, that ConA - inhibitor of clathrin mediated endocytosis - also abrogates GET but to a lesser extent, and when using Dynasore - reversible inhibitor of dynamin - there is no effect on GET efficacy, if endocytosis is blocked for only 5min after GET. Moreover, MβCD also reduced GET efficacy in vivo in m. tibialis cranialis and this effect was long lasting. The results of this study show that endocytosis is probably the main mechanism of entrance of DNA after GET in vitro and also in vivo.
    Bioelectrochemistry 08/2014; 103. DOI:10.1016/j.bioelechem.2014.08.020 · 4.17 Impact Factor
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    • "One of the therapeutic genes that has already demonstrated its radio-sensitizing effect is IL-12 [44-52]. Although not fully elucidated, proposed mechanisms of IL-12 radio-sensitization were enhanced tumor antigen presentation due to radiation induced apoptosis [49], anti-angiogenic effects [42], and the production of radiosensitizer nitric oxide [52]. In addition to potent local radio-sensitizing activity [44-52], IL-12 gene therapy can also offer the systemic protection against distant metastases by induction of an effective immune response against tumor antigens or inhibiting angiogenesis of metastases [21,24,25,53]. "
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    ABSTRACT: Radiation induced transcriptional targeting is a gene therapy approach that takes advantage of the targeting abilities of radiotherapy by using radio inducible promoters to spatially and temporally limit the transgene expression. Cyclin dependent kinase inhibitor 1 (CDKN1A), also known as p21, is a crucial regulator of the cell cycle, mediating G1 phase arrest in response to a variety of stress stimuli, including DNA damaging agents like irradiation. The aim of the study was to evaluate the suitability of the p21 promoter for radiation induced transcriptional targeting with the objective to test the therapeutic effectiveness of the combined radio-gene therapy with p21 promoter driven therapeutic gene interleukin 12. To test the inducibility of the p21 promoter, three reporter gene experimental models with green fluorescent protein (GFP) under the control of p21 promoter were established by gene electrotransfer of plasmid DNA: stably transfected cells, stably transfected tumors, and transiently transfected muscles. Induction of reporter gene expression after irradiation was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging using fluorescence stereomicroscope in vivo. The antitumor effect of the plasmid encoding the p21 promoter driven interleukin 12 after radio-gene therapy was determined by tumor growth delay assay and by quantification of intratumoral and serum levels of interleukin 12 protein and intratumoral concentrations of interleukin 12 mRNA. Using the reporter gene experimental models, p21 promoter was proven to be inducible with radiation, the induction was not dose dependent, and it could be re-induced. Furthermore radio-gene therapy with interleukin 12 under control of the p21 promoter had a good antitumor therapeutic effect with the statistically relevant tumor growth delay, which was comparable to that of the same therapy using a constitutive promoter. In this study p21 promoter was proven to be a suitable candidate for radiation induced transcriptional targeting. As a proof of principle the therapeutic value was demonstrated with the radio-inducible interleukin 12 plasmid providing a synergistic antitumor effect to radiotherapy alone, which makes this approach feasible for the combined treatment with radiotherapy.
    Molecular Cancer 11/2013; 12(1):136. DOI:10.1186/1476-4598-12-136 · 4.26 Impact Factor
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