Agustín Díaz

Nanoencapsulation of Insulin into Zirconium Phosphate for Oral Delivery Applications

The encapsulation of insulin into different kinds of materials for noninvasive delivery is an important field of study because of the many drawbacks of painful needle and syringe delivery such as physiological stress, infection, and local hypertrophy, among others (Khafagy, E.-S.; et al. Adv. Drug Delivery Rev. 2007, 59 (15), 1521-1546). A stable, robust, nontoxic, and viable noninvasive carrier for insulin delivery is needed. We present a new approach for protein nanoencapsulation using layered zirconium phosphate (ZrP) nanoparticles produced without any preintercalator present. The use of ZrP without preintercalators produces a highly pure material, without any kinds of contaminants, such as the preintercalator, which can be noxious. Cytotoxicity cell viability in vitro experiments for the ZrP nanoparticles show that ZrP is not toxic, or harmful, in a biological environment, as previously reported for rats (Zhu, Z. Y.; et al. Mater. Sci. Forum 2009, 620-622, 307-310). Contrary to previous preintercalator-based methods, we show that insulin can be nanoencapsulated in ZrP if a highly hydrate phase of ZrP with an interlayer distance of 10.3 Å (10.3 Å-ZrP or θ-ZrP) is used as a precursor. The intercalation of insulin into ZrP produced a new insulin-intercalated ZrP phase with about a 27 A interlayer distance, as determined by X-ray powder diffraction, demonstrating a successful nanoencapsulation of the hormone. The in vitro release profile of the hormone after the intercalation was determined and circular dichroism was used to study the hormone stability upon intercalation and release. The insulin remains stable in the layered material, at room temperature, for a considerable amount of time, improving the shell life of the peptidic hormone. This type of material represents a strong candidate to developing a noninvasive insulin carrier for the treatment of diabetes mellitus.

Pickering emulsions stabilized by amphiphilic nano-sheets

We demonstrate the fabrication of amphiphilic nano-sheets, which are either surface- or edge-modified plates with thickness at the atomic scale, one of the thinnest amphiphilic particles reported so far. They are obtained by exfoliation of functionalized layered crystals, the first time that laminar structures have been utilized to produce such particles. Stable emulsions were made utilizing these nano-sheets. The adsorption of the amphiphilic nano-sheets onto the oil-in-water interfaces and the reduction of surface tension between the PDMS and the amphiphilic nano-sheet suspensions were quantitatively characterized.

Surface Functionalization of Zirconium Phosphate Nanoplatelets for the Design of Polymer Fillers

Inorganic-organic hybrid materials were synthesized by covalent attachment of epoxides to the surface of zirconium phosphate (ZrP) nanoplatelets. X-ray powder diffraction, FTIR, and TGA were utilized to confirm the presence of the modifiers and exclusive functionalization of the ZrP surface. NMR experiments were conducted to confirm the formation of P-O-C bonds between surface phosphate groups and epoxide rings. The applicability of the organically modified products was demonstrated by their use as fillers in a polymer matrix. Subsequently, a two step intercalation and surface modification procedure was utilized to prepare polymer nanocomposites that were imparted with functionality through the encapsulation of molecules within the interlayer of surface modified ZrP.

Nitrosyl hydride (HNO) as an O2 analogue: long-lived HNO-adducts of ferrous globins

Nitrosyl hydride, HNO or nitroxyl, is the one-electron reduced and protonated form of nitric oxide. HNO is isoelectronic to singlet O(2), and we have previously reported that deoxymyoglobin traps free HNO to form a stable adduct. In this report, we demonstrate that oxygen-binding hemoglobins from human, soy, and clam also trap HNO to form adducts which are stable over a period of weeks. The same species can be formed in higher yields by careful reduction of the ferrous nitrosyl adducts of the proteins. Like the analogous O(2)-Fe(II) adducts, the HNO adducts are diamagnetic, but with a characteristic HNO resonance in (1)H NMR at ca. 15 ppm that splits into doublets for H(15)NO adducts. The (1)H and (15)N NMR resonances, obtained by HSQC experiments, are shown to differentiate subunits and isoforms of proteins within mixtures. An apparent difference in the reduction rates of the NO adducts of the two subunits of human hemoglobin allows assignment of two distinct nitrosyl hydride peaks by a combination of UV-vis, NMR, and EPR analysis. The two peaks of the HNO-hHb adduct have a persistent 3:1 ratio during trapping reactions, demonstrating a kinetic difference between HNO binding at the two subunits. These results show NMR characterization of ferrous HNO adducts as a unique tool sensitive to structural changes within the oxygen-binding cavity, which may be of use in defining modes of oxygen binding in other heme proteins and enzymes.

Direct intercalation of cisplatin into zirconium phosphate nanoplatelets for potential cancer nanotherapy

We report the use of zirconium phosphate (ZrP) nanoplatelets for the encapsulation of the anticancer drug cisplatin and its delivery to tumor cells. Cisplatin was intercalated into ZrP by direct ion exchange and was tested in vitro for cytotoxicity in the human breast cancer (MCF-7) cell line. The structural characterization of the intercalated cisplatin in ZrP suggests that during the intercalation process, the chloride ligands of the cisplatin complex were substituted by phosphate groups within the layers. Consequently, a new phosphate phase with the platinum complex directly bound to ZrP (cisPt@ZrP) is produced with an interlayer distance of 9.3 Å. The in vitro release profile of the intercalated drug upon a pH stimulus shows that at low pH under lysosomal conditions the platinum complex is released with simultaneous hydrolysis of the zirconium phosphate material, while at higher pH the complex is not released. Experiments with the MCF-7 cell line show that cisPt@ZrP reduced the cell viability up to 40%. The cisPt@ZrP intercalation product is envisioned as a future nanotherapy agent against cancer. Taking advantage of the shape and sizes of the ZrP particles and controlled release of the drug at low pH, it is intended to exploit the enhanced permeability and retention effect of tumors, as well as their intrinsic acidity, for the destruction of malignant cells.

Designable Architectures on Nanoparticle Surfaces: Zirconium Phosphate Nanoplatelets as a Platform for Tetravalent Metal and Phosphonic Acid Assemblies

Surface-functionalized zirconium phosphate (ZrP) nanoparticles were synthesized using a combination of ion exchange and self-assembly techniques. The surface of ZrP was used as a platform to deposit tetravalent metal ions by direct ion exchange with the protons of the surface phosphate groups. Subsequently, phosphonic acids were attached to the metal ion layer, effectively functionalizing the ZrP nanoparticles. Use of axially oriented bisphosphonic acids led to the ability to build layer-by-layer assemblies from the nanoparticle surface. Varying the metal ion and ligand used allowed designable architectures to be synthesized on the nanoparticle surface. X-ray powder diffraction, XPS, electron microprobe, solid-state NMR, FTIR, and TGA were used to characterize the synthesized materials.

Abstract 2909: Doxorubicin-loaded zirconium phosphate nanoparticles for intracellular anticancer drug delivery to breast cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: Doxorubicin (DOX), is an anthracycline antibiotic widely used for the treatment of several solid tumors but its usage is limited by serious side effects to healthy tissues. The purpose of this study was to explore the effectiveness of zirconium phosphate nanoparticles (ZrP) as carrier of anticancer agents (ie. DOX) to tumor cells and potentially reduce side effects to healthy tissue. Methods: Confocal Microscopy (CM) was used to assess internalization and localization of doxorubicin-loaded ZrP nanoparticles (DOX:ZrP) in MCF-7 breast cancer cell line. The cytotoxicity of ZrP and DOX:ZrP in MCF-7 and MCF-10A cells was studied using MTT assay. Results: Co-internalization of DOX and ZrP nanoparticles into the cytoplasm and nucleus of MCF-7 cells was detected using CM. A high fluorescence in cell nucleus demonstrated DOX release from ZrP nanoparticles. ZrP alone is non-toxic to MCF-7 and MCF-10A cells. After a 48-hour treatment of MCF-7 cells with DOX:ZrP or DOX alone the IC50 obtained was 23µM and 24 µM, respectively. DOX cytotoxicity to MCF-7 cells after 72-hour treatment was three times higher than DOX:ZrP with IC50 of 0.74 µM and 3.15 µM, respectively. Conclusion: ZrP nanoparticles are internalized and can effectively deliver DOX to the interior of cancer cells. ZrP nanoparticles might provide a versatile platform for transportation of anticancer agents resulting in improved cancer therapy by reducing the side effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2909. doi:1538-7445.AM2012-2909

Abstract 5512: Zirconium phosphate nanoparticle matrix as potential delivery system for cisplatin to cancer cells in vitro

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cisplatin (cis-diamminedichloroplatinum(II), have been shown to be active against ovarian and testicular cancer, Hodgkins lymphoma, and certain other malignancies, however, its clinical effectiveness is limited by significant side effects and the emergence of drug resistance. Delivery of anticancer drugs such as cisplatin to the tumor cells without damaging healthy organs or tissues is highly difficult if not impossible. In the last several years nanoparticle-mediated drug delivery is being studied as a novel valuable mean for overcoming this problem. We developed a novel nanoparticle intercalated formulation of cisplatin on a layered matrix of zirconium phosphate (ZrP). It was expected that cisplatin, while being excluded from the external medium within ZrP layers will remain biologically inactive and that cisplatin will be released selectively in the tumor cell. The purpose of this study was to determine the cytotoxicity and effectiveness of ZrP nanoparticles in the delivery of cisplatin. Cell viability was measured by MTT assay. Flow cytometry analysis was used to determine cisplatin-induced apoptosis, and cell cycle distribution. ZrP alone did not affect the viability of breast cancer cells (MCF-7), human leukemic lymphoblast (CEM) and human peripheral blood lymphocytes (PBLs) after 24 hours in contact with different concentrations of ZrP. Cisplatin intercalated into ZrP at the highest concentration (30 μM cisplatin) reduced in 40% the cell viability of MCF-7 and CEM. The IC50 of cisplatin alone in MCF-7 was 10 μM. The side scatter plot showed that normal PBLs scatter plot is different from PBLs that have been in contact with cisplatin-intercalated ZrP for 18 hours. These PBLs carrying ZrP were displaced to the right quadrant suggesting an increased granularity that can be associated to the incorporation of ZrP nanoparticles. Moderate cisplatin-induced apoptosis was evident in cells treated with ZrP-intercalated cisplatin for 18 hours. The cell cycle analysis showed that ZrP alone does not cause cell cycle arrest. Our results suggest that application of ZrP nanoparticle-mediated drug transport in cancer chemotherapy is highly promising and more studies are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5512.