Tumor Targeting by Monoclonal Antibody Functionalized Magnetic Nanoparticles

Tumor-targeted drug-loaded nanocarriers represent innovative and attractive tools for cancer therapy. Several magnetic nanoparticles (MNPs) were analyzed as potential tumor-targeted drug-loaded nanocarriers after functionalization with anti-Met oncogene (anti-Met/HGFR) monoclonal antibody (mAb) and doxorubicin (DOXO). Their cytocompatibility, stability, immunocompetence (immunoprecipitation), and their interactions with cancer cells in vitro (Perl’s staining, confocal microscopy, cytotoxic assays: MTT, real time toxicity) and with tumors in vivo (Perl’s staining) were evaluated. The simplest silica- and calcium-free mAb-loaded MNPs were the most cytocompatible, the most stable, and showed the best immunocompetence and specificity. These mAb-functionalized MNPs specifically interacted with the surface of Met/HGFR-positive cells, and not with Met/HGFR-negative cells; they were not internalized, but they discharged in the targeted cells DOXO, which reached the nucleus, exerting cytotoxicity. The presence of mAbs on DOXO-MNPs significantly increased their cytotoxicity on Met/HGFR-positive cells, while no such effect was detectable on Met/HGFR-negative cells. Bare MNPs were biocompatible in vivo; mAb presence on MNPs induced a better dispersion within the tumor mass when injected in situ in Met/HGFR-positive xenotumors in NOD/SCID-y null mice. These MNPs may represent a new and promising carrier for in vivo targeted drug delivery, in which applied gradient and alternating magnetic fields can enhance targeting and induce hyperthermia respectively.This research was funded by AIRC—Italy, grant number IG n. 13166, Compagnia di San Paolo, grant CSP-Torino-Piemonte: 12-CSP-C04-018, and the Università del Piemonte Orientale “A. Avogadro”, grant 02

Tumor targeting by monoclonal antibody functionalized magnetic nanoparticles

Tumor-targeted drug-loaded nanocarriers represent innovative and attractive tools for cancer therapy. Several magnetic nanoparticles (MNPs) were analyzed as potential tumor-targeted drug-loaded nanocarriers after functionalization with anti-Met oncogene (anti-Met/HGFR) monoclonal antibody (mAb) and doxorubicin (DOXO). Their cytocompatibility, stability, immunocompetence (immunoprecipitation), and their interactions with cancer cells in vitro (Perl's staining, confocal microscopy, cytotoxic assays: MTT, real time toxicity) and with tumors in vivo (Perl's staining) were evaluated. The simplest silica- and calcium-free mAb-loaded MNPs were the most cytocompatible, the most stable, and showed the best immunocompetence and specificity. These mAb-functionalized MNPs specifically interacted with the surface of Met/HGFR-positive cells, and not with Met/HGFR-negative cells; they were not internalized, but they discharged in the targeted cells DOXO, which reached the nucleus, exerting cytotoxicity. The presence of mAbs on DOXO-MNPs significantly increased their cytotoxicity on Met/HGFR-positive cells, while no such effect was detectable on Met/HGFR-negative cells. Bare MNPs were biocompatible in vivo; mAb presence on MNPs induced a better dispersion within the tumor mass when injected in situ in Met/HGFR-positive xenotumors in NOD/SCID-γnull mice. These MNPs may represent a new and promising carrier for in vivo targeted drug delivery, in which applied gradient and alternating magnetic fields can enhance targeting and induce hyperthermia respectively

Eu-Doped Citrate-Coated Carbonated Apatite Luminescent Nanoprobes for Drug Delivery

In the field of Nanomedicine, there is an increasing demand for new inorganic nanophosphors with low cytotoxicity and efficient loading-release ability of drugs for applications in bioimaging and drug delivery. This work assesses the potentiality of matured Eu-doped citrate-coated carbonated apatite nanoparticles to be used as theranostic platforms, for bioimaging, as luminescent nanoprobes, and for drug delivery applications, using Doxorubicin as a model drug. The drug adsorption isotherm fits the Langmuir–Freundlich (LF) model, showing that the Eu:cit-cAp nanoparticles can carry a maximum of 0.29 +/- 0.02 mg Doxo mg Eu:cit-cAp-1 (Qmax). The affinity constant KFL for this binding is 44 +/- 2 mL mg-1, and the cooperativity coefficient r is 6 +/- 1. The nanoparticle suspensions presented charge reversion from negative to positive after loading with Doxo as revealed by the c-potential versus pH characterization. The release of drug from the loaded nanoparticles was found to be strongly pH-dependent, being around 5 wt % at physiological pH 7.4 and 20 wt % at pH 5, in experiments lasting 24 h. Luminescence spectroscopic measurements of Doxo-loaded nanoparticles revealed the increase of luminescence with a decrease in the amount of adsorbed Doxo, due to the so-called inner filter effect. The nanoparticles free of Doxo were cytocompatible when interacted with two human cell lines derived respectively from a gastric carcinoma (GTL-16), and a hepatocarcinoma (Huh7), while Doxo-loaded nanoparticles displayed significant toxicity in a dose-dependent relationship. Therefore, the new nanoassemblies might have a dual function, as nanoprobes in bioimaging by detecting the fate of the nanoparticles in biological environments, and for monitoring the delivery of the drug in such environments, by measuring the rise of the luminescence provided by the desorption of Doxo.This research was funded by Spanish Agencia Estatal de Investigación of the Ministerio de Ciencia, Innovación y Universidades and co-funded with FEDER, UE, Project No. PGC2018-102047-B-I00 (MCIU/AEI/FEDER, UE). The APC was funded by Grant No. PGC2018-102047-B-I00 (MCIU/AEI/FEDER, UE). C.J.-L. thanks project CGL2016-76723 (MINECO/FEDER, UE). Y.J. wants to acknowledge an FPU2016 grant (Ref. FPU16_04580)

Bioinspired Mineralization of Type I Collagen Fibrils with Apatite in Presence of Citrate and Europium Ions

Synthetic nanostructured hybrid composites based on collagen and nanocrystalline apatites are interesting materials for the generation of scaffolds for bone tissue engineering. In this work, mineralized collagen fibrils were prepared in the presence of citrate and Eu3+. Citrate is an indispensable and essential structural/functional component of bone. Eu3+ endows the mineralized fibrils of the necessary luminescent features to be potentially employed as a diagnostic tool in biomedical applications. The assembly and mineralization of collagen were performed by the neutralization method, which consists in adding dropwise a Ca(OH)2 solution to a H3PO4 solution containing the dispersed type I collagen until neutralization. In the absence of citrate, the resultant collagen fibrils were mineralized with nanocrystalline apatites. When citrate was added in the titrant solution in a Citrate/Ca molar ratio of 2 or 1, it acted as an inhibitor of the transformation of amorphous calcium phosphate (ACP) to nanocrystalline apatite. The addition of Eu3+ and citrate in the same titrant solution lead to the formation of Eu3+–doped citrate–coated ACP/collagen fibrils. Interestingly, the relative luminescent intensity and luminescence lifetime of this latter composite were superior to those of Eu3+–doped apatite/collagen prepared in absence of citrate. The cytocompatibility tests, evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) colorimetric assay in a dose–dependent manner on GTL–16 human gastric carcinoma cells, on MG–63 human osteosarcoma cells and on the m17.ASC, a spontaneously immortalized mouse mesenchymal stem cell clone from subcutaneous adipose tissue, show that, in general, all samples are highly cytocompatible.This research was funded by Spanish MINEICO and co–funded by FEDER (grant number MAT2014–60533–R). C. V.–E. acknowledges the Spanish MINEICO for his contract PTA2015–11103–I. M.I. acknowledges support by the Italian National Research Program–National Research Council (PNRCNR) Aging Program 2012–2014. The Excellence Network of Crystallography and Crystallization “Factoría de Cristalización” FIS2015–71928–REDC funded by Spanish MINEICO is also acknowledged

Monophasic and Biphasic Electrical Stimulation Induces a Precardiac Differentiation in Progenitor Cells Isolated from Human Heart

Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α(1G)) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype

A sustainable one-pot method to transform seashell waste calcium carbonate to osteoinductive hydroxyapatite micro-nanoparticles

We have developed a straightforward, one-pot, low-temperature hydrothermal method to transform oyster shell waste particles (bCCP) from the species Crassostrea gigas (Mg-calcite, 5 wt% Mg) into hydroxyapatite (HA) micro/nanoparticles. The influence of the P reagents (H3PO4, KH2PO4, and K2HPO4), P/bCCP molar ratios (0.24, 0.6, and 0.96), digestion temperatures (25-200 & DEG;C), and digestion times (1 week-2 months) on the transformation process was thoroughly investigated. At 1 week, the minimum temperature to yield the full transformation significantly reduced from 160 & DEG;C to 120 & DEG;C when using K2HPO4 instead of KH2PO4 at a P/bCCP ratio of 0.6, and even to 80 & DEG;C at a P/bCCP ratio of 0.96. The transformation took place via a dissolution-reprecipitation mechanism driven by the favorable balance between HA precipitation and bCCP dissolution, due to the lower solubility product of HA than that of calcite at any of the tested temperatures. Both the bCCP and the derived HA particles were cytocompatible for MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells, and additionally, they promoted the osteogenic differentiation of m17.ASC, especially the HA particles. Because of their physicochemical features and biological compatibility, both particles could be useful osteoinductive platforms for translational applications in bone tissue engineering

Bioinspired crystallization, sensitized luminescence and cytocompatibility of citrate-functionalized Ca-substituted europium phosphate monohydrate nanophosphors

Bio compatible nanosystems exhibiting long lifetime ( ~millisecond) luminescence features are particu l arly relevant in the field of bioimaging. In this study, citrate functionalized calcium doped europium phosphates nanophosphors of the rhabdophane type were prepared at different synthesis times by a bioinspired crystallization route, consisting in thermal decomplexing of ca2•tEu3• {citrate{phosphate{car bonate solutions. The general formula of this material is Ca«Eu1 a(PO4) 1 a(HP04 l,,•nH2O, with CJ. ranging from 0 to 0.58 and n ~ 1. A thorough characterization of the nanoparticles has been carried out by XRD (including data processing with Topas 6.0), HR TEM, TEM, FTIR, TG{ITTA. ICP, dynamic light scattering (OLS), electrophoretic mobility, and fluorescence spectroscopy. Based on these results a crystallization mechanism involving the filling of cationic sites with Ca21ons associated to a concomitant adjustment of the P04{HPO4 ratio was proposed Upon calcium doping, the aspect ratio of the nanoparticles as well as of the crystalline domains decreased and the relative luminescence intensity (R.LI.) could be modulated. Neither the pH nor the ionic strength, nor the temperature (from 25 to 37 C) affected signif icantly the R.L.I. of particles after resuspension in water, leading to rather steady luminescence features usable in a large domain of conditions. This new class of luminescent compounds has been proved to be fully cytocompatible relative to GTL 16 human carcinoma cells and showed an improved cytocompatibil ity as the Ca2+ content increased when contacted with the more sensitive m17. ASC murine mesenchymal stem cells. These biocompatible nanoparticles thus appear as promising new tailorable tools for biomed ical applications as luminescent nanoprobes.We greatly acknowledge the project Biomin nanoapatite MAT2014 60533 R supported by Spanish MINEICO and co funded by FEDER and the Excellence Network of Crystallography and Crystallization "Factoría de Cristalización" FIS2015 71928 REDC supported by Spanish MINEICO. Cristóbal Verdugo Escamilla also acknowledges the Spanish MINEICO for his contract PT A2015 11103 I

Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol

Correction: Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

[This corrects the article DOI: 10.1371/journal.pone.0137999.]

Luminescent biomimetic citrate-coated europium-doped carbonated apatite nanoparticles for use in bioimaging: physico-chemistry and cytocompatibility

Nanomedicine covers the application of nanotechnologies in medicine. Of particular interest is the setup of highly-cytocompatible nanoparticles for use as drug carriers and/or for medical imaging. In this context, luminescent nanoparticles are appealing nanodevices with great potential for imaging of tumor or other targetable cells, and several strategies are under investigation. Biomimetic apatite nanoparticles represent candidates of choice in nanomedicine due to their high intrinsic biocompatibility and to the highly accommodative properties of the apatite structure, allowing many ionic substitutions. In this work, the preparation of biomimetic (bone-like) citrate-coated carbonated apatite nanoparticles doped with europium ions is explored using the citrate-based thermal decomplexing approach. The technique allows the preparation of the single apatitic phase with nanosized dimensions only at Eu3+ doping concentrations ≤0.01 M at some timepoints. The presence of the citrate coating on the particle surface (as found in bone nanoapatites) and Eu3+ substituting Ca2+ is beneficial for the preparation of stable suspensions at physiological pH, as witnessed by the ζ-potential versus pH characterizations. The sensitized luminescence features of the solid particles, as a function of the Eu3+ doping concentrations and the maturation times, have been thoroughly investigated, while those of particles in suspensions have been investigated at different pHs, ionic strengths and temperatures. Their cytocompatibility is illustrated in vitro on two selected cell types, the GTL-16 human carcinoma cells and the m17.ASC murine mesenchymal stem cells. This contribution shows the potentiality of the thermal decomplexing method for the setup of luminescent biomimetic apatite nanoprobes with controlled features for use in bioimaging