Jun 25, 2010 - Magnetite-APTES Nps were synthesized using a simple and cheap ... [i] Lee J. H., Huh Y. M., Jun Y. W., Seo J. W., Jang J. T., Song H. T., Kim S.
Study of highly compatible nanostructured systems for drug delivery and medical diagnostics Ph.D. Thesis summary Doctorate in Chemical Sciences (XXIII cycle) Student: Nepi Fabio Thesis project aims are based on the unexpected properties of NPs (NPs), in particular magnetic nanosystems, with the purpose of having a high level multifunctional system for the following main objectives: ●
Drug delivery
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Medical diagnostics
The first intention, drug delivery, is possible because of the magnetic susceptibility of our material, that could be driven in a tissue as well as in an organism by means of an external magnetic field; the magnetic system could so be focused into the desired zone to improve the effectiveness of the drug that they are carryingi. Drug delivery system is allowed by anchoring organic molecules on the surface of the magnetic particle, in order to bind bioactive drugs. As a matter of fact, this system could couple a local biochemical treatment with a thermal shock by means of an alternating magnetic field applied after that the delivery system has been localized in the interested area (thermoablation)ii,iii,iv. The second main objective is based on the opportunity to detect a magnetic nucleus in an aqueous matrix using a 1H-MRI (1H-Magnetic Resonance Imaging) apparatus. Indeed, as it is known by literaturev,vi magnetic colloids can modify relaxation times (T1, T2, T2*), so it is possible to detect magnetic NPs in tissues detecting magnetic distortions. As it will be shown below, the use of magnetic systems improves the sensibility of MRI and allows us to detect low concentration magnetoparticles, increasing the signal-to-noise ratio and, as a consequence, having important applications in the field of MR medical diagnosis and multifunctional devices (such as PET-MRI instruments). Since the magnetic properties are very important for our scopes, we synthesized 2 different structured magnetic NPs, in order to explore their different behaviour. The systems are: ●
Fe3O4-APTES
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Fe@Cu@Au
The first system uses an oxidized iron magnetic nucleus coated with a modified siloxane (APTES – 3-aminopropyl-triethoxy silane), the second one uses a zero-valent magnetic nucleus covered with 2 metallic shells, respectively copper and gold; magnetic nucleus and the copper shell are useful for the PET-MRI diagnostic technique (since we can use 64Cu salts for the covering process and so having a magnetic-positron emitting nanometric material); gold shell is useful for the linkage of organic molecules with the aim of binding bioactive molecules, since it can easily bond molecules with functional groups like thiols or disulphides; in such situation we could have a nanoshaped system that we can, at the same time, use as a drug delivery system and follow it by means of PETMRI instruments. Our material was synthesized using wet methods in aqueous solutions, with cheap reactants. All these systems were characterized by means of XRPD (X-Ray Powder Diffraction), AFM (Atomic Force Microscopy), VSM (Vibrating Sample Magnetometer), in order to determine the crystal structure, the diameter of the particles, the magnetic properties. Fe3O4-APTES, in addition, was analyzed using FT-IR (Fourier Transform-InfraRed spectroscopy) and XPS (X-ray Photoemission Spectroscopy) to determine the linkage of APTES on the magnetic NP. The synthesis of Fe@Cu@Au NPs was based on a three-step method: the precipitation and thickening of metal copper on the Fe nucleus, and the formation of a new gold shell. The main goal of the process is the use of a zero-valent iron nucleus both as seeding for the growing of the metallic copper shell, and as for reducing agent for the precipitation of metallic copper from ionic one. The covering process with gold is allowed by the direct reaction with the metallic copper present in the outer shell of the first-step NP(Fe@Cu). This innovative synthetic method is very useful, simple and let us to consider the synthetic process as a new instrument for the build-up of nanostructured systems. Structural characterization showed that our NPs have a high crystalline structure, and morphological analysis (AFM) suggested us that the product have a final size of about 50 nm. Fe@Cu@Au NPs were then modified using cystamine, in order to link a fluorescent dye (fluoresceine isothiocyanate), to prove the cellular engulfment. After the coating our system was characterized via FT-IR to detect the surface links, in addition this material was internalized in human cells to prove their biocompatibility and their fluorescent skills. Magnetite-APTES Nps were synthesized using a simple and cheap two-step reaction developed in our laboratory. The first step based on a fast and cheap co-precipitation of Fe3O4 in aqueous solution was the synthesis of the magnetic nucleus, improving and optimizing the method
proposed by Massartvii,viii. The second step was the coating reaction with APTES, according to a Stöber reaction, successfully modified in our laboratoryix. Since the final experiment was the internalization of our NPs in human cells (mixed human leukocytes) and their visualization in MRI experiments, we linked on our nanosystems a fluorescent dye (FITC - Fluorescein IsoThioCyanate), to prove the effective engulfment of our systems by cells. As-synthesized NPs were characterized in order to detect crystallinity, size and linking of the organic molecule (XRPD, AFM, FT-IR). This synthesis method allowed us to collect fluorescent microscopy images and to prove the internalization of labelled NPs in human cells. Fluorescent micrographs showed that the engulfment has taken place, ant the cells were still live after the incubation overnight with the modified NPs. MRI images for our two systems were collected in T2*; the images showed a high contrast and good relaxation parameters (T1, T2, T2*); these data prove the possibility to use our NPs as contrast agent in MRI, since we obtained high-contrast images for low concentration of NPs, in addition, these structure showed an important biocompatibility, due to the evidence that our engulfed cells were alive after the internalization.
[i] Lee J. H., Huh Y. M., Jun Y. W., Seo J. W., Jang J. T., Song H. T., Kim S., Cho E. J., Yoon H. G., Suh J. S., Cheon J., Nat. Med. 2007, 13, p. 95; [ii] Mitsumori M., Hiraoka M., Shibata T., Okuno Y., Nagata Y., Nishimura Y., Abe M., Hasegawa M., Nagae H., Ebisawa Y., Hepato-Gastroenterology, 1996, 43, p. 1431; [iii] Lïubbe A. S., Bergemann C., Huhnt W., Fricke T., Riess H., Brock J., W., Huhn D. Cancer Res., 1996, 56, p. 4694; [iv] Viroonchatapan E., Ueno M., Sato H., Adachi I., Nagae H., Tazawa K., Horikoshi I., Pharmaceut. Res., 1995, 12, p. 1176; [v] Yoon T. J., Kim J. S., Kim B. G., Yu K. N., Cho M. H., Lee J. K., Angew. Chem., Int. Ed., 2005, 44, p. 1068; [vi] Kim D. K., Zhang Y., Voit W., Rao K. V., Kehr J., Bjelke B., Muhammed M., Scr. Mater., 2001 44, p. 1713; [vii] Buske N., Gotze T., Sonntag H., Colloids Surf. 1984, 12, p. 195; [viii] Goetze T., Gansau C., Buske N., Roeder M., G.ornert P., Bahr M., Journal of magnetism and magnetic materials, 2002, 252, p. 399;
[ix] de Almeida M.P.S., Caiado K.L., Sartoratto P.P.C., Cintra e Silva D.O., Pereira A.R., Morais P.C., Journal of Alloys and Compounds, 25 June 2010, 500, (2), p. 149;
