The Role of Polymeric Coatings for a Safe-by-Design Development of Biomedical Gold Nanoparticles Assessed in Zebrafish Embryo.

In the biomedical field, gold nanoparticles (GNPs) have attracted the attention of the scientific community thanks to their high potential in both diagnostic and therapeutic applications. The extensive use of GNPs led researchers to investigate their toxicity, identifying stability, size, shape, and surface charge as key properties determining their impact on biological systems, with possible strategies defined to reduce it according to a Safe-by-Design (SbD) approach. The purpose of the present work was to analyze the toxicity of GNPs of various sizes and with different coating polymers on the developing vertebrate model, zebrafish. In particular, increasing concentrations (from 0.001 to 1 nM) of 6 or 15 nm poly-(isobutylene-alt-maleic anhydride)-graft-dodecyl polymer (PMA)- or polyethylene glycol (PEG)-coated GNPs were tested on zebrafish embryos using the fish embryo test (FET). While GNP@PMA did not exert significant toxicity on zebrafish embryos, GNP@PEG induced a significant inhibition of embryo viability, a delay of hatching (with the smaller size NPs), and a higher incidence of malformations, in terms of tail morphology and eye development. Transmission electron microscope analysis evidenced that the more negatively charged GNP@PMA was sequestered by the positive charges of chorion proteins, with a consequent reduction in the amount of NPs able to reach the developing embryo and exert toxicological activity. The mild toxic response observed on embryos directly exposed to GNP@PMA suggest that these NPs are promising in terms of SbD development of gold-based biomedical nanodevices. On the other hand, the almost neutral GNP@PEG, which did not interact with the chorion surface and was free to cross chorion pores, significantly impacted the developing zebrafish. The present study raises concerns about the safety of PEGylated gold nanoparticles and contributes to the debated issue of the free use of this nanotool in medicine and nano-biotechnologies.

Mn(II)-Based Lipidic Nanovesicles as High-Efficiency MRI Probes.

Although nowadays there is a renewed and growing interest in Mn-based contrast agents, there are only few studies dealing with Mn-based lipophilic nanoparticles and how they may be optimized as MRI contrast agents. Three amphiphilic paramagnetic Mn(II) complexes based on derivatives of EDTA and 1,4-DO2A were used for the preparation of lipidic nanoparticles. The length and position of the aliphatic chains were found to control whether either vesicular liposomes, nonvesicular bicelles, or a mixture of both was produced as well as the size and morphology of phospholipid-based self-assembling nanoaggregates. These differences determine whether hydrophilic Gd-based contrast agents or fluorescent dyes can be entrapped in the aqueous core of the nanoaggregate. Structural characterization was performed by cryo-TEM. Detailed 1H NMR relaxometric analyses were carried out on all systems. The effect of entrapping gadoteridol in the aqueous core (where present) was studied by preparing diamagnetic amphiphilic Zn(II) analogues. In the case of homogeneous systems, the data were also fitted to obtain the relaxometric parameters for comparison with literature data. The results of these studies demonstrate enhanced relaxivity of the nanoaggregates with respect to monomeric analogues. This work allowed us to understand how to control the formation of different types of nanovesicles (liposomes, bicelles, and micelles), optimize their MRI contrast, and provide different in vivo biodistribution characteristics.

Supramolecular assemblies based on amphiphilic Mn2+-complexes as high relaxivity MRI probes.

In the research field of MRI contrast agents (CAs), amphiphilic paramagnetic complexes are typically sought for the increased plasmatic half-life and high relaxivity values, but limited examples of amphiphilic Mn2+-based CAs have been reported to date. In this work the Mn2+-complexes of six original amphiphilic ligands (three EDTA-like ligands and three 1,4-DO2A derivatives) embodying one or two aliphatic chains were evaluated as potential MRI contrast agents and compared. Strong self-association into micelles resulted in a relaxivity (r1) enhancement (ca. 80% with respect to MnEDTA) as a consequence of the increased molecular tumbling rate of the supramolecular aggregate. In the case of bis-substituted systems the r1 gain is much higher due to the restricted local rotation of the chelates about the pendant aliphatic chains (r1 in the range 12.6-18.4 mM-1 s-1, 2-3 times higher than for the micelles obtained with single-chain EDTA systems). Furthermore, these amphiphilic chelates tightly bind to human serum albumin (HSA) with association constants KA in the range 104-105 M-1. The resulting supramolecular adducts achieve remarkable relaxivity values, in the range 50-60 mM-1 s-1 for the MnEDTA-like chelates and 27-30 mM-1 s-1 for the 1,4-DO2A-like systems (at 298 K and 20 MHz), thanks to their fast water exchange rate.

High relaxivity Mn(2+)-based MRI contrast agents.

Stable Mn(2+) mono- and binuclear complexes containing pentadentate 6,6'-((methylazanediyl)bis(methylene))dipicolinic acid coordinating units give remarkably high relaxivities due to the presence of two inner-sphere water molecules. The mononuclear derivative binds human serum albumin (HSA) with an association constant of 3372 M(-1), which results in the replacement of the coordinated water molecules by donor atoms of protein residues. The dinuclear analogue also binds HSA while leaving one of the Mn(2+) centres exposed to the solvent with two coordinated water molecules. Thus, this complex shows remarkably high relaxivities upon protein binding (39.0 mM(-1) s(-1) per Mn, at 20 MHz and 37 °C).

Picolinate-containing macrocyclic Mn2+ complexes as potential MRI contrast agents.

We report the synthesis of the ligand Hnompa (6-((1,4,7-triazacyclononan-1-yl)methyl)picolinic acid) and a detailed characterization of the Mn(2+) complexes formed by this ligand and the related ligands Hdompa (6-((1,4,7,10-tetraazacyclododecan-1-yl)methyl)picolinic acid) and Htempa (6-((1,4,8,11-tetraazacyclotetradecan-1-yl)methyl)picolinic acid). These ligands form thermodynamically stable complexes in aqueous solution with stability constants of logKMnL = 10.28(1) (nompa), 14.48(1) (dompa), and 12.53(1) (tempa). A detailed study of the dissociation kinetics of these Mn(2+) complexes indicates that the decomplexation reaction at about neutral pH occurs mainly following a spontaneous dissociation mechanism. The X-ray structure of [Mn2(nompa)2(H2O)2](ClO4)2 shows that the Mn(2+) ion is seven-coordinate in the solid state, being directly bound to five donor atoms of the ligand, the oxygen atom of a coordinated water molecule and an oxygen atom of a neighboring nompa(-) ligand acting as a bridging bidentate carboxylate group (µ-η(1)-carboxylate). Nuclear magnetic relaxation dispersion ((1)H NMRD) profiles and (17)O NMR chemical shifts and transverse relaxation rates of aqueous solutions of [Mn(nompa)](+) indicate that the Mn(2+) ion is six-coordinate in solution by the pentadentate ligand and one inner-sphere water molecule. The analysis of the (1)H NMRD and (17)O NMR data provides a very high water exchange rate of the inner-sphere water molecule (kex(298) = 2.8 × 10(9) s(-1)) and an unusually high value of the (17)O hyperfine coupling constant of the coordinated water molecule (AO/ℏ = 73.3 ± 0.6 rad s(-1)). DFT calculations performed on the [Mn(nompa)(H2O)](+)·2H2O system (TPSSh model) provide a AO/ℏ value in excellent agreement with the one obtained experimentally.

Comprehensive evaluation of the physicochemical properties of Ln(III) complexes of aminoethyl-DO3A as pH-responsive T(1) -MRI contrast agents.

N-Substituted aminoethyl groups were attached to 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) with the aim to design pH-responsive Ln(III) complexes based on the pH-dependent on/off ligation of the amine nitrogen to the metal ion. The following ligands were synthesized: AE-DO3A (aminoethyl-DO3A), MAE-DO3A (N-methylaminoethyl-DO3A), DMAE-DO3A (N,N-dimethylaminoethyl-DO3A) and MEM-AE-DO3A (N-methoxyethyl-N-methylaminoethyl-DO3A). The physicochemical properties of the Ln(III) complexes were investigated for the evaluation of their potential applicability as magnetic resonance imaging (MRI) contrast agents. In particular, a (1) H and (17) O NMR relaxometric study was carried out for these Gd(III) complexes at two different pH values: at basic pH (pendant amino group coordinated to the metal centre) and at acidic pH (protonated amine, not interacting with the metal ion). Eu(III) complexes allow one to estimate the number of inner-sphere water molecules through luminescence lifetime measurements and obtain some structural information through variable-temperature (VT) high-resolution (1) H NMR studies. Equilibria between differently hydrated species were found for most of the complexes at both acidic and basic pH. The thermodynamic stability of Ca(II) , Zn(II) , Cu(II) and Ln(III) complexes and kinetics of formation and dissociation reactions of Ln(III) complexes of AE-DO3A and DMAE-DO3A were investigated showing stabilities comparable to currently approved Gd(III) -based CAs. In detail, higher total basicity (Σlog Ki (H) ) and higher stability constants of Ln(III) complexes were found for AE-DO3A with respect to DMAE-DO3A (i.e., log KGd-AE-DO3A =22.40 and log KGd-DMAE-DO3A =20.56). The transmetallation reactions of Gd(III) complexes are very slow (Gd-AE-DO3A: t1/2 =2.7×10(4)  h; Gd-DMAE-DO3A: 1.1×10(5)  h at pH 7.4 and 298 K) and occur through proton-assisted dissociation.

Hyperfine coupling constants on inner-sphere water molecules of a triazacyclononane-based Mn(II) complex and related systems relevant as MRI contrast agents.

We report the synthesis of the ligand H2MeNO2A (1,4-bis(carboxymethyl)-7-methyl-1,4,7-triazacyclononane) and a detailed experimental and computational study of the hyperfine coupling constants (HFCCs) on the inner-sphere water molecules of [Mn(MeNO2A)] and related Mn(2+) complexes relevant as potential contrast agents in magnetic resonance imaging (MRI). Nuclear magnetic relaxation dispersion (NMRD) profiles, (17)O NMR chemical shifts, and transverse relaxation rates of aqueous solutions of [Mn(MeNO2A)] were recorded to determine the parameters governing the relaxivity in this complex and the (17)O and (1)H HFCCs. DFT calculations (TPSSh model) performed in aqueous solution (PCM model) on the [Mn(MeNO2A)(H2O)]·xH2O and [Mn(EDTA)(H2O)](2-)·xH2O (x = 0-4) systems were used to determine theoretically the (17)O and (1)H HFCCs responsible for the (17)O NMR chemical shifts and the scalar contributions to (17)O and (1)H NMR relaxation rates. The use of a mixed cluster/continuum approach with the explicit inclusion of a few second-sphere water molecules is critical for an accurate calculation of HFCCs of coordinated water molecules. The impact of complex dynamics on the calculated HFCCs was evaluated with the use of molecular dynamics simulations within the atom-centered density matrix propagation (ADMP) approach. The (17)O and (1)H HFCCs calculated for these complexes and related systems show an excellent agreement with the experimental data. Both the (1)H and (17)O HFCCs (A(iso) values) are dominated by the spin delocalization mechanism. The A(iso) values are significantly affected by the distance between the oxygen atom of the coordinated water molecule and the Mn(2+) ion, as well as by the orientation of the water molecule plane with respect to the Mn-O vector.

Comparative in vitro studies of MR imaging probes for metabotropic glutamate subtype-5 receptor targeting.

A series of magnetic resonance imaging probes has been evaluated to target selectively the metabotropic glutamate receptor subtype 5 (mGluR5). Eight imaging probes based on the contrast agent [Gd·DOTA], previously derived by linking it to a series of specific and selective mGluR5 antagonists, have been extensively tested for their functionality in vitro. The Nuclear Magnetic Relaxation Dispersion (NMRD) profiles of selected probes have been examined via field-cycling relaxometry in the presence and absence of a model protein. The properties of the targeted contrast agents were evaluated using a primary astrocyte model, as these cells mimic the in vivo situation effectively. The probes were non-toxic (up to 200 µM) to these mGluR5 expressing primary cells. Cellular proton longitudinal relaxation rate enhancements of up to 35% were observed by MRI at 200 µM of probe concentration. The antagonistic effect of all compounds was tested using an assay measuring changes of intracellular calcium levels. Furthermore, treatment at two different temperatures (4 °C vs. 37 °C) and of an mGluR5-negative cell line provided further insight into the selectivity and specificity of these probes towards cell surface mGluR5. Finally, two out of eight probes demonstrated an antagonistic effect as well as significant enhancement of receptor mediated cellular relaxation rates, strongly suggesting that they would be viable probes for the mapping of mGluR5 by MRI in vivo.

Scaling Laws at the Nano Size: The Effect of Particle Size and Shape on the Magnetism and Relaxivity of Iron Oxide Nanoparticle Contrast Agents.

The magnetic properties of iron oxide nanoparticles govern their relaxivities and efficacy as contrast agents for MRI. These properties are in turn determined by their composition, size and morphology. Herein we present a systematic study of the effect of particle size and shape of magnetite nanocrystals synthesized by thermal decompositions of iron salts on both their magnetism and their longitudinal and transverse relaxivities, r1 and r2, respectively. Faceted nanoparticles demonstrate superior magnetism and relaxivities than spherical nanoparticles of similar size. For faceted nanoparticles, but not for spherical ones, r1 and r2 further increase with increasing particle size up to a size of 18 nm. This observation is in accordance with increasing saturation magnetization for nanoparticles increasing in size up to 12 nm, above which a plateau is observed. The NMRD (Nuclear Magnetic Resonance Dispersion) profiles of MIONs (Magnetic Iron Oxide Nanoparticles) display an increase in longitudinal relaxivity with decreasing magnetic field strength with a plateau below 1 MHz. The transverse relaxivity shows no dependence on the magnetic field strength between 20 MHz and 500 MHz. These observations translate to phantom MR images: in T1-weighted SWIFT (SWeep imaging with Fourier Transform) images MIONs have a positive contrast with little dependence on particle size, whereas in T2-weighted gradient-echo images MIONs create a negative contrast which increases in magnitude with increasing particle size. Altogether, these results will enable the development of particulate MRI contrast agents with enhanced efficacy for biomedical and clinical applications.

Self-aggregated dinuclear lanthanide(III) complexes as potential bimodal probes for magnetic resonance and optical imaging.

Homodinuclear lanthanide complexes (Ln = La, Eu, Gd, Tb, Yb and Lu) derived from a bis-macrocyclic ligand featuring two 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid chelating sites linked by a 2,6-bis(pyrazol-1-yl)pyridine spacer (H2L(3)) were prepared and characterized. Luminescence lifetime measurements recorded on solutions of the Eu(III) and Tb(III) complexes indicate the presence of one inner-sphere water molecule coordinated to each metal ion in these complexes. The overall luminescence quantum yields were determined (ϕ H2O = 0.01 for [Eu2(L(3))] and 0.50 for [Tb2(L(3))] in 0.01 M TRIS/HCl, pH 7.4; TRIS = tris(hydroxymethyl)aminomethane), pointing to an effective sensitization of the metal ion by the bispyrazolylpyridyl unit of the ligand, especially with Tb. The nuclear magnetic relaxation dispersion (NMRD) profiles recorded for [Gd2(L(3))] are characteristic of slowly tumbling systems, showing a low-field plateau and a broad maximum around 30 MHz. This suggests the occurrence of aggregation of the complexes giving rise to slowly rotating species. A similar behavior is observed for the analogous Gd(III) complex containing a 4,4'-dimethyl-2,2'-bipyridyl spacer ([Gd2(L(1))]). The relaxivity of [Gd2(L(3))] recorded at 0.5 T and 298 K (pH 6.9) amounts to 13.7 mM(-1) s(-1). The formation of aggregates has been confirmed by dynamic light scattering (DLS) experiments, which provided mean particle sizes of 114 and 38 nm for [Gd2(L(1))] and [Gd2(L(3))], respectively. TEM images of [Gd2(L(3))] indicate the formation of nearly spherical nanosized aggregates with a mean diameter of about 41 nm, together with some nonspherical particles with larger size.

Paramagnetic solid lipid nanoparticles as a novel platform for the development of molecular MRI probes.

Highly efficient magnetic resonance imaging (MRI) probes have been prepared by loading Gd(III) complexes on the surface of solid lipid nanoparticles (pSLNs). Applicability as molecular imaging probes is demonstrated by an in vitro model study with targeted pSLNs.

1H and 17O NMR relaxometric and computational study on macrocyclic Mn(II) complexes.

Herein we report a detailed 1H and 17O relaxometric investigation of Mn(II) complexes with cyclen-based ligands such as 2-(1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (DO1A), 2,2'-(1,4,7,10-tetraazacyclododecane-1,4-diyl)diacetic acid (1,4-DO2A), 2,2'-(1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid (1,7-DO2A), and 2,2',2"-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DO3A). The Mn(II) complex with the heptadentate ligand DO3A does not have inner sphere water molecules (q = 0), and therefore, the metal ion is most likely seven-coordinate. The hexadentate DO2A ligand has two isomeric forms: 1,7-DO2A and 1,4-DO2A. The Mn(II) complex with 1,7-DO2A is predominantly six-coordinate (q = 0). In aqueous solutions of [Mn(1,4-DO2A)], a species with one coordinated water molecule (q = 1) prevails largely, whereas a q = 0 form represents only about 10% of the overall population. The Mn(II) complex of the pentadentate ligand DO1A also contains a coordinated water molecule. DFT calculations (B3LYP model) are used to obtain information about the structure of this family of closely related complexes in solution, as well as to determine theoretically the 17O and 1H hyperfine coupling constants responsible for the scalar contribution to 17O and 1H NMR relaxation rates and 17O NMR chemical shifts. These calculations provide 17O A/h values of ca. 40 × 10(6) rad s(-1), in good agreement with experimental data. The [Mn(1,4-DO2A)(H2O)] complex is endowed with a relatively fast water exchange rate (k(ex)298 = 11.3 × 10(8) s(-1)) in comparison to the [Mn(EDTA)(H2O)]2- analogue (k(ex)298 = 4.7 × 10(8) s(-1)), but about 5 times lower than that of the [Mn(DO1A)(H2O)]+ complex (k(ex)298 = 60 × 10(8) s(-1)). The water exchange rate measured for the latter complex represents the highest water exchange rate ever measured for a Mn(II) complex.

Orthogonal synthesis of a heterodimeric ligand for the development of the Gd(III)-Ga(III) ditopic complex as a potential pH-sensitive MRI/PET probe.

A heterodimeric polyaminocarboxylate ligand based on a DO3A-sulfonamide linked to AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetracarboxylic acid) was synthesised via an orthogonal pathway in order to differentiate the two chelating cages and allow the formation of a Gd(III)-Ga(III) heteroditopic complex. The goal is to create a smart MRI/PET probe with pH dependent relaxivity and with the bimodal imaging approach that enables direct quantification of the stimulus, in this case pH. A (1)H NMR relaxometric study of the Gd-Ga heteroditopic complex addressed the pH modulation of the relaxivity and thus its possible use as an MRI pH sensitive probe.

One-pot synthesis of a piperidine-based rigidified DTPA analogue and its bifunctional chelating agent.

The core structure of cis-3,5-diaminopiperidine was N-alkylated with excess t-butylbromoacetate in order to exploit the successive N-quaternarization and Stevens rearrangement to access the pentaalkylated product and the bifunctional chelating agent containing a N-butanedioic acid pendant arm at the same time. The relaxometric properties of the Gd(III) complexes with these ligands were studied also in terms of pH and serum stabilities.

Responsive Mn(II) complexes for potential applications in diagnostic Magnetic Resonance Imaging.

The investigation of new Mn(II)-based MRI/Molecular Imaging probes responsive to the enzyme tyrosinase for potential diagnostic applications is herein described. The expression of the enzyme tyrosinase, an oxidoreductase, is up-regulated in melanoma cancer cells. Three novel ligands (L(1), L(2) and L(3)) were designed as modified acyclic polyaminocarboxylate chelates by introducing an l-tyrosine residue in place of an aminoacetate unit. The corresponding Mn(II) complexes were fully characterised by (1)H NMR relaxometric techniques in aqueous media. The responsive activity towards the expression of tyrosinase was then assessed by monitoring the (1)H 1/T(1) relaxivity changes during incubation experiments in buffered solutions containing tyrosinase at different concentrations and in B16F10 melanoma cell homogenate. New insight on the mechanism of action of these systems was gained by measuring the magnetic field dependence of the relaxivity and ESR spectra of the incubated solutions. The systems developed showed responsive activity to tyrosinase with a relaxation enhancement spanning from 50% (MnL(1)) to 350% (MnL(3)) which augurs well for the development of diagnostic probes to detect melanoma cancer.

Studies on the synthesis of the ABC rings of (+/-)-hexacyclinic acid.

A synthesis of the ABC-rings of the polyketide natural product hexacyclinic acid has been achieved. The B-ring was formed first via an intramolecular ester-tethered Diels-Alder reaction, and the A-ring was annulated to this by means of a SmI(2) mediated reductive 5-exo-trig cyclization of a samarium-ketyl radical onto a vinyl group. Finally, the C-ring was closed using olefin metathesis. Interestingly, use of enyne metathesis resulted in the synthesis of a more functionalized 5-membered C-ring in a model system, but an undesired 6-membered C-ring in the actual system. An investigation of this change in selectivity is discussed.