Multifunctional zirconium phosphate layered materials for the nanodelivery of anticancer drugs

Abstract

In this thesis work, we addressed the use of the inorganic layered nanomaterial (ILN) zirconium bis(monohydrogen orthophosphate) monohydrate (Zr(HPO₄)·H₂O, α-ZrP) as a nanocarrier for the delivery of doxorubicin and cisplatin anticancer drugs.<br /> <br /> Cancer has been the second cause of death in the United States since the 1950s. Since then, many efforts have been made to overcome the limitations of current anticancer treatments. The application of nanotechnology to the delivery of chemotherapeutics is a highly investigated area with promising results. The encapsulation of anticancer drugs into nanocarriers and their surface modification has been reported to significantly improve their pharmacokinetics properties, inducing a selective biodistribution and targeting tumor tissue, improving retention times and biocompatibility. The induced selectivity and improved biocompatibility contribute to the reduction of adverse side effects, drug resistance, and administrated dose.<br /> <br /> The synthesis and characterization of the ZrP as a precursor for the nanocarrier were investigated over time. The α-layered structure and nanoparticle formation were monitored at 1, 5, 24, and 48 hours of reaction following Kijima's method for synthesizing the hydrated θ-phase of ZrP. X-ray powder diffraction (XRPD) patterns obtained for θ-ZrP and the α-ZrP phase produced by dehydration of the former showed the expected interlayer distances of 10.4 Å and 7.6 Å at all reaction times, respectively. However, small nanoparticles (up to 50 nm) of very low crystallinity were formed at 1 and 5 hours of reaction, as confirmed by XRPD results and transmission electron microscopy (TEM) images. After 24 hours of synthesis, the crystallinity of the ZrP samples was significantly improved, and hexagonal shape nanoparticles of ca. 200 nm were observed employing TEM and dynamic light scattering (DLS) techniques. ³¹P-nuclear magnetic resonance (³¹P-NMR) results showed the characteristic phosphorous resonance at ca. -19 ppm corresponding to P-OH groups in the interlayer space at all reaction times. In comparison, a higher concentration of dehydrated regions was observed in the samples obtained at 1 and 5 hours of reaction confirmed by a phosphorous resonance at -21 ppm. The presence of dehydrated areas was confirmed with infrared spectroscopy (IR). From these results, we conclude that the α-like structure of ZrP is formed at the early stages of synthesis. The nanoparticles aging process involves an increment in aspect ratio, possibly due to the fusion of smaller nanoparticles and the incorporation of water molecules in the interlayer space.<br /> <br /> The intercalation of DOX into ZrP nanoparticles and the surface modification of the intercalated material with Zr(IV) and monomethoxy-polyethylene glycol-monophosphate (PEG) molecules were performed to improve the nanocarrier properties of ZrP. The successful intercalation of DOX into ZrP produced DOX@ZrP materials with interlayer distances from 19 to 22 Å as a function of the molar ratio in agreement with the DOX dimensions. The surface modification of DOX@ZrP was monitored by XRPD and NMR. After the two steps of surface modification with Zr(IV) and PEG no changes in the DOX@ZrP interlayer distance were observed, which confirms that the functionalization was restricted to the surface. The presence of PEG was confirmed by thermogravimetric analysis and ¹³C-NMR. The presence of a new phosphorous environment after the surface modification with Zr(IV) was confirmed by ³¹P-NMR with a new resonance at -27 ppm. A downfield shift of this signal was observed after the surface modification with PEG, suggesting the formation of a chemical bond between phosphate groups from PEG molecules and Zr(IV) metals on the surface of DOX@ZrP. The viability and internalization of the surface-modified products were evaluated in human prostate cancer (PC3) cells, showing highly reduced cell viability and improved internalization capability of PEG/Zr/DOX@ZrP materials compared with the non-PEGylated ones.<br /> <br /> The combined intercalation of two positively charged molecules, with remarkable differences in size and structure, into layered ZrP nanoplatelets by direct ion exchange was also performed. DOX and CisPt were intercalated using three different methods: one-step simultaneous intercalation of both molecules (OSI) and two-step intercalation (TSI-A for the intercalation of DOX in the first step and TSI-B when CisPt is intercalated first). Although elemental analysis data was obtained using inductively coupled plasma-optical emission spectroscopy (ICP-OES) showed Pt loading of ca. 10% regardless of the intercalation method, scanning/transmission electron microscopy (S/TEM) images, and XRPD results demonstrated remarkable differences between both intercalation methods. The OSI method produces a material that segregates cations into different layers. The TSI-A method results in a random distribution of cations in all layers, suggesting that combined intercalation occurred in the same interlayer space. On the other hand, TSI-B samples demonstrated that DOX could not intercalate when CisPt is intercalated first. Extensive structural characterization was performed using TGA analysis, solid-states ³¹P-NMR, and FT-IR spectroscopy. The viability of ovarian resistance cancer (OVCAR3CIS) cells was evaluated against OSI, TSI-A, TSI-B and compared the individually intercalated materials DOX@ZrP and CisPt@ZrP and their physical mixture, and the individual and combined unintercalated drugs after 24 and 48 hours of exposure. OSI and TSI-A showed high toxicity compared to TSI-B and behave similarly to DOX@ZrP, suggesting that the cytotoxic effects is due mainly to the presence of DOX. This study adds to the comprehension of the cointercalation chemistry of ZrP materials and their applications to cancer nanotherapy.