For application of nanoparticles (NPs) as delivery vectors for biomedical applications the size, charge, surfac chemistry and aggregation of the particles are one of key parameters. We focused on characterization of maghemite and cobalt ferrite magnetic NPs by measuring size distribution and zeta potential in different culture media and PBS. We show that our NPs functionalized with PAA are relatively very stable also in different culture media, where level of aggregation depends on medium composition. Effect of divalent ions and serum presence on stability is specifically analysed and we present possible destabilization mechanisms. We show that stability of electrostatically stabilized suspensions is affected by the molar concentration and valence of destabilizing counterions like Na+ or Mg2+ where effective surface charge of nanoparticles and thus repulsion force is screened by these counterions. In agreement with other papers we demonstrate significant effect of media composition (divalent ions, serum) and thus show the importance of NPs characterization under conditions that are representative of cell culture media or physiological conditions for understanding of NPs interaction with biological systems and for assessments of nanoparticle cytotoxicity. Further, by understanding the destabilization mechanisms one can anticipate effect in different media and to some degree predict behaviour of nanoparticle suspensions.
COBISS.SI-ID: 9593172
Several internalization and cytotoxicity studies have been performed, but there are still many unanswered questions concerning the interactions of nanoparticles and cells. We prepared functionalized magnetic NPs, coated with polyacrylic acid (PAA), which are stable also in physiological conditions and short-term non-toxic. Using scanning and transmission electron microscopy, we were able to observe and determine the internalization pathways in Chinese hamster ovary (CHO) cells. We could observe that NP were internalized as small aggregates via macropinocytosis and clathrin mediated endocytosis, resulting in rapid uptake in a time-dependent manner. Inside the cytoplasm, aggregated NPs were found enclosed in acidified vesicles accumulated in the perinuclear region 1 h after exposure, where they stayed for at least up to 24 h. Moreover, using scanning electron microscopy we also captured what might be the first step of NPs internalization - an endocytic vesicle in the process of formation enclosing NPs bound to the membrane. Since no cytotoxic effects were observed, the NDs are potentially suitable for biomedical applications (e.g. labeling). In preparation is international patent application.
COBISS.SI-ID: 30476761
We have developed a 3D in vitro model of cells embedded in collagen gels, which represent a complex system similar to realistic environment in tissues. Cells in 3D gel are viable and anable analysis of electrotransfection or magentofection. 3D collagen models are very important since th emobility of nanoparticles or macromolecules is similar to mobility in tissues, which enables design and optimization of protocols for magnetofection in a more realistic in vitro model. Gel models represent important step toward clinical trials, since they reduce the number of sacrificed animals.
COBISS.SI-ID: 7803220
We developed and characterized a new type of magnetic nanoparticle delivery system for gene transfer, with option of further functionalization for imaging or drug delivery. In combination, appropriate magnetic system for magnetofection (gene transfer with help of magnetic field) were designed and developed. These nanoparticles are optimized for intake in cell in in vitro conditions with application of external magnetic fields and exhibit increased uptake in cells with relatively low cytotoxicity. Such nanoparticles are very appropriate vector system for uptake of active components in cells and can be applied in biotechnological and biomedical applications like magnetofection. The results are used in collaboration with Institute of Oncology for development of in vitro magnetofection and further translation of the method to in vivo systems. Magnetofection of cells with plasmid DNA encoding reportergene using our magnetic NDs was superior in transfection efficiency to commercially available nanoparticles.
COBISS.SI-ID: 967035
The the type of magnetic nanoparticles developed for magnetofections in course of the project lead by PI have been further developed as delivery system for gene transfer in vivo for cancer therapy. Appropriate magnetic system for magnetofection (gene transfer with help of magnetic field) were designed and developed. In collaboration with Institute of Oncology we have sucesfully achieved significant antitumor effect in vivo of murine adenocarcinoma using our magnetic nanoparticles functionalized with DNA encoding interleukin IL – 12, which can be futher used for immuno-gene therapy.
COBISS.SI-ID: 1237883