optimized-methods-for-surface-immobilization-collagens-collagen

Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ ...

Université d'Orléans

Metal complexes are increasingly explored as imaging probes in amyloid peptide related pathologies. We report the first detailed study on the mechanism of interaction between a metal complex and both the monomer and the aggregated form of Aβ1-40 peptide. We have studied lanthanide(III) chelates of two PiB-derivative ligands (PiB=Pittsburgh compound B), L(1) and L(2), differing in the length of the spacer between the metal-complexing DO3A macrocycle (DO3A=1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) and the peptide-recognition PiB moiety. Surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR spectroscopy revealed that they both bind to aggregated Aβ1-40 (KD =67-160 μM), primarily through the benzothiazole unit. HSQC NMR spectroscopy on the (15) N-labeled, monomer Aβ1-40 peptide indicates nonsignificant interaction with monomeric Aβ. Time-dependent circular dichroism (CD), dynamic light scattering (DLS), and TEM investigations of the secondary structure and of the aggregation of Aβ1-40 in the presence of increasing amounts of the metal complexes provide coherent data showing that, despite their structural similarity, the two complexes affect Aβ fibril formation distinctly. Whereas GdL(1), at higher concentrations, stabilizes β-sheets, GdL(2) prevents aggregation by promoting α-helical structures. These results give insight into the behavior of amyloid-targeted metal complexes in general and contribute to a more rational design of metal-based diagnostic and therapeutic agents for amyloid- associated pathologies.

Interaction of PiB-derivative metal complexes with beta-amyloid peptides: selective recognition of the aggregated forms.

We report first prototypes of responsive lanthanide(III) complexes that can be monitored independently in three complementary imaging modalities. Through the appropriate choice of lanthanide(III) cations, the same reactive ligand can be used to form complexes providing detection by (i) visible (Tb(3+)) and near-infrared (Yb(3+)) luminescence, (ii) PARACEST- (Tb(3+), Yb(3+)), or (iii) T1-weighted (Gd(3+)) MRI. The use of lanthanide(III) ions of different natures for these imaging modalities induces only a minor change in the structure of complexes that are therefore expected to have a single biodistribution and cytotoxicity.

Prototypes of Lanthanide(III) Agents Responsive to Enzymatic Activities in Three Complementary Imaging Modalities: Visible/Near-Infrared Luminescence, PARACEST-, and T1-MRI.

Lanthanide complexes have attracted much attention in the biomedical field, and today various imaging applications make use of their versatile magnetic and luminescence properties. In this minireview, we give insight into the mechanistic aspects that allow modulation of the relaxation or chemical exchange saturation transfer (CEST) features, and thus the magnetic resonance imaging (MRI) efficiency of paramagnetic lanthanide chelates in order to create agents that are capable of providing an MRI response as a function of a specific biomarker. We focus on the detection of neurotransmitters, enzymatic activities, and amyloid peptides. We also describe two selected theranostic strategies: 1) a novel approach directed at monitoring drug release from liponanoparticles and 2) molecular or nanoparticle probes for the MRI visualization of photosensitizer delivery in photodynamic therapy.

Lanthanide Complexes in Molecular Magnetic Resonance Imaging and Theranostics.

Aggregation of misfolded proteins and progressive polymerization of otherwise soluble proteins is a common hallmark of a wide range of highly debilitating and increasingly prevalent diseases, including amyotrophic lateral sclerosis, cerebral amyloid angiopathy, type II diabetes and Parkinson's, Huntington's and Alzheimer's diseases. There is a growing interest in creating imaging agents to detect such aggregates in various imaging modalities, including PET, SPECT and MRI. We present here an overview of recent efforts from the perspective of early diagnosis of amyloid diseases, with a major focus on Aβ detection and metal complexes bearing PiB units.

Metal complexes for multimodal imaging of misfolded protein-related diseases.

We propose quantitative assessment of zinc by combining nuclear and MR imaging. We use a cocktail of a Gd3+-complex providing a Zn2+-dependent MRI response and its 165Er3+ analogue allowing for concentration assessment. 165Er is readily obtained in a cyclotron and purified, which is indispensable for successful quantification of metal ions.

A cocktail of Er(iii) and Gd(iii) complexes for quantitative detection of zinc using SPECT and MRI.

Gold nanoparticles (AuNPs) are interesting for the design of new cancer theranostic tools, mainly due to their biocompatibility, easy molecular vectorization, and good biological half-life. Herein, we report a gold nanoparticle platform as a bimodal imaging probe, capable of coordinating Gd for Magnetic Resonance Imaging (MRI) and Ga for Single Photon Emission Computed Tomography (SPECT) imaging. Our AuNPs carry a bombesin analogue with affinity towards the gastrin releasing peptide receptor (GRPr), overexpressed in a variety of human cancer cells, namely PC3 prostate cancer cells. The potential of these multimodal imaging nanoconstructs was thoroughly investigated by the assessment of their magnetic properties, in vitro cellular uptake, biodistribution, and radiosensitisation assays. The relaxometric properties predict a potential T- and T- MRI application. The promising in vitro cellular uptake of Ga/Gd-based bombesin containing particles was confirmed through biodistribution studies in tumor bearing mice, indicating their integrity and ability to target the GRPr. Radiosensitization studies revealed the therapeutic potential of the nanoparticles. Moreover, the DOTA chelating unit moiety versatility gives a high theranostic potential through the coordination of other therapeutically interesting radiometals. Altogether, our nanoparticles are interesting nanomaterial for theranostic application and as bimodal T- and T- MRI / SPECT imaging probes.

Dual Imaging Gold Nanoplatforms for Targeted Radiotheranostics.

The search for more biocompatible alternatives to Gd -based MRI agents, and the interest in  Mn for PET imaging call for ligands that form inert Mn chelates. Given the labile nature of Mn , high inertness is challenging to achieve. The strongly preorganized structure of the 2,4-pyridyl-disubstituted bispidol ligand L endows its Mn complex with exceptional kinetic inertness. Indeed, MnL did not show any dissociation for 140 days in the presence of 50 equiv. of Zn (37 °C, pH 6), while recently reported potential MRI agents MnPyC3A and MnPC2A-EA have dissociation half-lives of 0.285 h and 54.4 h under similar conditions. In addition, the relaxivity of MnL (4.28 mm  s at 25 °C, 20 MHz) is remarkable for a monohydrated, small Mn chelate. In vivo MRI experiments in mice and determination of the tissue Mn content evidence rapid renal clearance of MnL . Additionally, L could be radiolabeled with  Mn and the complex revealed good stability in biological media.

Unprecedented Kinetic Inertness for a Mn -Bispidine Chelate: A Novel Structural Entry for Mn -Based Imaging Agents.

Metal chelates targeted to amyloid peptides are widely explored as diagnostic tools or therapeutic agents. The attachment of a metal complex to amyloid recognition units typically leads to a decrease in peptide affinity. We show here that by separating a macrocyclic GdL chelate and a PiB targeting unit with a long hydrophobic C10 linker, it is possible to attain nanomolar affinities for both Aβ (K =4.4 nm) and amylin (K =4.5 nm), implicated, respectively in Alzheimer's disease and diabetes. The Scatchard analysis of surface plasmon resonance data obtained for a series of amphiphilic, PiB derivative GdL complexes indicate that their Aβ or amylin binding affinity varies with their concentration, thus micellar aggregation state. The GdL chelates also affect peptide aggregation kinetics, as probed by thioflavin-T fluorescence assays. A 2D NMR study allowed identifying that the hydrophilic region of Aβ is involved in the interaction between the monomer peptide and the Gd complex. Finally, ex vivo biodistribution experiments were conducted in healthy mice by using  In labeled analogues. Their pancreatic uptake, ∼3 %ID g , is promising to envisage amylin imaging in diabetic animals.

Concentration-Dependent Interactions of Amphiphilic PiB Derivative Metal Complexes with Amyloid Peptides Aβ and Amylin*.

Invited for the cover of this issue are Jean-François Morfin and Éva Tóth at the CNRS in Orléans, and their collaborators from University of Debrecen, University of Coimbra and Université de Toulouse. The image depicts that when an amphiphilic compound is intravenously injected, monomer, pre-micellar and micellar forms can co-exist in the blood and have different affinities for amyloid peptides. Read the full text of the article at 10.1002/chem.202004000.

Drug-loaded liposomes are typical examples of nanomedicines. We show here that doxorubicin, the anti-cancer agent in the liposomal drug Doxil, can sensitize Ytterbium (Yb ) and generate its near-infrared (NIR) emission. When doxorubicin and amphiphilic Yb chelates are incorporated into liposomes, the sensitized emission of Yb is dependent on the integrity of the particles, which can be used to monitor drug release. We also established the first demonstration that the NIR Yb emission signal is observable in living mice following intratumoral injection of the Yb -doxorubicin-liposomes, using a commercial macroscopic setup equipped with a NIR camera.

Doxorubicin-Sensitized Luminescence of NIR-Emitting Ytterbium Liposomes: Towards Direct Monitoring of Drug Release.

Dysfunctional elastin turnover plays a major role in the progression of atherosclerotic plaques. Failure of tropoelastin cross-linking into mature elastin leads to the accumulation of tropoelastin within the growing plaque, increasing its instability. Here we present Gd-TESMA, an MRI contrast agent specifically designed for molecular imaging of tropoelastin within plaques. Gd-TESMA is a tetrameric probe composed of a tropoelastin-binding peptide (the VVGS-peptide) conjugated with four Gd(III)-DOTA-monoamide chelates. It shows a relaxivity per molecule of 34.0 ± 0.8 mM s (20 MHz, 298 K, pH 7.2), a good binding affinity to tropoelastin ( = 41 ± 12 μM), and a serum half-life longer than 2 h. Gd-TESMA accumulates specifically in atherosclerotic plaques in the ApoE murine model of plaque progression, with 2 h persistence of contrast enhancement. As compared to the monomeric counterpart (Gd-TESMA), the tetrameric Gd-TESMA probe shows a clear advantage regarding both sensitivity and imaging time window, allowing for a better characterization of atherosclerotic plaques.

Imaging of Dysfunctional Elastogenesis in Atherosclerosis Using an Improved Gadolinium-Based Tetrameric MRI Probe Targeted to Tropoelastin.

Porous materials, such as zeolites, have great potential for biomedical applications, thanks to their ability to accommodate positively charged metal-ions and their facile surface functionalization. Although the latter aspect is important to endow the nanoparticles with chemical/colloidal stability and desired biological properties, the possibility for simple ion-exchange enables easy switching between imaging modalities and/or combination with therapy, depending on the envisioned application. In this study, the nanozeolite Linde type L (LTL) with already confirmed magnetic resonance imaging properties, generated by the paramagnetic gadolinium (Gd) in the inner cavities, was successfully radiolabeled with a positron emission tomography (PET)-tracer zirconium-89 (Zr). Thereby, exploiting Zr-chloride resulted in a slightly higher radiolabeling in the inner cavities compared to the commonly used Zr-oxalate, which apparently remained on the surface of LTL. Intravenous injection of PEGylated Zr/Gd-LTL in healthy mice allowed for PET-computed tomography evaluation, revealing initial lung uptake followed by gradual migration of LTL to the liver and spleen. Ex vivo biodistribution confirmed the in vivo stability and integrity of the proposed multimodal probe by demonstrating the original metal/Si ratio being preserved in the organs. These findings reveal beneficial biological behavior of the nanozeolite LTL and hence open the door for follow-up theranostic studies by exploiting the immense variety of metal-based radioisotopes.

On the Versatility of Nanozeolite Linde Type L for Biomedical Applications: Zirconium-89 Radiolabeling and In Vivo Positron Emission Tomography Study.

Bispidine (3,7-diazabicyclo[3.3.1]nonane) provides a rigid and preorganized scaffold that is particularly interesting for the stable and inert complexation of metal ions, especially for their application in medical imaging. In this study, we present the synthesis of two bispidine ligands with -methanephosphonate (H) and -methanecarboxylate (H) substituents as well as the physico-chemical properties of the corresponding Mn and Zn complexes. The two complexes [Mn()] and [Mn()] have relatively moderate thermodynamic stability constants according to potentiometric titration data. However, they both display an exceptional kinetic inertness, as assessed by transmetallation experiments in the presence of 50 equiv excess of Zn, showing only ∼40 and 20% of dissociation for [Mn()] and [Mn()], respectively, after 150 days at pH 6 and 37 °C. Proton relaxivities amount to  = 4.31 mM s ([Mn()]) and 3.64 mM s ([Mn()]) at 20 MHz, 25 °C, and are remarkable for Mn complexes with one inner-sphere water molecule ( = 1); they are comparable to that of the commercial contrast agent [Gd(DOTA)(HO)]. The presence of one inner-sphere water molecule and an associative water exchange mechanism was confirmed by temperature-dependent transverse O relaxation rate measurements, which yielded  = 0.12 × 10 and 5.5 × 10 s for the water exchange rate of the phosphonate and the carboxylate complex, respectively. In addition, radiolabeling experiments with Mn were also performed with H() showing excellent radiolabeling properties and quantitative complexation at pH 7 in 15 min at room temperature as well as excellent stability of the complex in various biological media over 24 h.

Article	Year
Interaction of PiB-derivative metal complexes with beta-amyloid peptides: selective recognition of the aggregated forms. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 25712142	2015
Prototypes of Lanthanide(III) Agents Responsive to Enzymatic Activities in Three Complementary Imaging Modalities: Visible/Near-Infrared Luminescence, PARACEST-, and T1-MRI. Journal of the American Chemical Society\| PubMed ID: 26727374	2016
Lanthanide Complexes in Molecular Magnetic Resonance Imaging and Theranostics. ChemMedChem\| PubMed ID: 28470934	2017
Metal complexes for multimodal imaging of misfolded protein-related diseases. Dalton transactions (Cambridge, England : 2003)\| PubMed ID: 28952628	2017
A cocktail of Er(iii) and Gd(iii) complexes for quantitative detection of zinc using SPECT and MRI. Chemical communications (Cambridge, England)\| PubMed ID: 29789839	2018
Dual Imaging Gold Nanoplatforms for Targeted Radiotheranostics. Materials (Basel, Switzerland)\| PubMed ID: 31978954	2020
Unprecedented Kinetic Inertness for a Mn -Bispidine Chelate: A Novel Structural Entry for Mn -Based Imaging Agents. Angewandte Chemie (International ed. in English)\| PubMed ID: 32298021	2020
Concentration-Dependent Interactions of Amphiphilic PiB Derivative Metal Complexes with Amyloid Peptides Aβ and Amylin*. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 33026686	2021
Concentration-Dependent Interactions of Amphiphilic PiB Derivative Metal Complexes with Amyloid Peptides Aβ and Amylin*. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 33258526	2021
Doxorubicin-Sensitized Luminescence of NIR-Emitting Ytterbium Liposomes: Towards Direct Monitoring of Drug Release. Angewandte Chemie (International ed. in English)\| PubMed ID: 34387934	2021
Imaging of Dysfunctional Elastogenesis in Atherosclerosis Using an Improved Gadolinium-Based Tetrameric MRI Probe Targeted to Tropoelastin. Journal of medicinal chemistry\| PubMed ID: 34661390	2021
On the Versatility of Nanozeolite Linde Type L for Biomedical Applications: Zirconium-89 Radiolabeling and In Vivo Positron Emission Tomography Study. ACS applied materials & interfaces\| PubMed ID: 35830285	2022
Mn Complexes with a Bispidine-Phosphonate Ligand: High Kinetic Inertness for Imaging Applications. Inorganic chemistry\| PubMed ID: 35984220	2022

Sara Lacerda

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