Effect of the Size and Shape of Dendronized Iron Oxide Nanoparticles Bearing a Targeting Ligand on MRI, Magnetic Hyperthermia, and Photothermia Properties-From Suspension to In Vitro Studies.

Functionalized iron oxide nanoparticles (IONPs) are increasingly being designed as a theranostic nanoplatform combining specific targeting, diagnosis by magnetic resonance imaging (MRI), and multimodal therapy by hyperthermia. The effect of the size and the shape of IONPs is of tremendous importance to develop theranostic nanoobjects displaying efficient MRI contrast agents and hyperthermia agent via the combination of magnetic hyperthermia (MH) and/or photothermia (PTT). Another key parameter is that the amount of accumulation of IONPs in cancerous cells is sufficiently high, which often requires the grafting of specific targeting ligands (TLs). Herein, IONPs with nanoplate and nanocube shapes, which are promising to combine magnetic hyperthermia (MH) and photothermia (PTT), were synthesized by the thermal decomposition method and coated with a designed dendron molecule to ensure their biocompatibility and colloidal stability in suspension. Then, the efficiency of these dendronized IONPs as contrast agents (CAs) for MRI and their ability to heat via MH or PTT were investigated. The 22 nm nanospheres and the 19 nm nanocubes presented the most promising theranostic properties (respectively, r2 = 416 s-1·mM-1, SARMH = 580 W·g-1, SARPTT = 800 W·g-1; and r2 = 407 s-1·mM-1, SARMH = 899 W·g-1, SARPTT = 300 W·g-1). MH experiments have proven that the heating power mainly originates from Brownian relaxation and that SAR values can remain high if IONPs are prealigned with a magnet. This raises hope that heating will maintain efficient even in a confined environment, such as in cells or in tumors. Preliminary in vitro MH and PTT experiments have shown the promising effect of the cubic shaped IONPs, even though the experiments should be repeated with an improved set-up. Finally, the grafting of a specific peptide (P22) as a TL for head and neck cancers (HNCs) has shown the positive impact of the TL to enhance IONP accumulation in cells.

Physical Chemistry Study of Collagen-Based Multilayer Films.

The surface properties of a biomaterial play an important role in cell behavior, e.g., recolonization, proliferation, and migration. Collagen is known to favor wound healing. In this study, collagen (COL)-based layer-by-layer (LbL) films were built using different macromolecules as a partner, i.e., tannic acid (TA), a natural polyphenol known to establish hydrogen bonds with protein, heparin (HEP), an anionic polysaccharide, and poly(sodium 4-styrene sulfonate) (PSS), an anionic synthetic polyelectrolyte. To cover the whole surface of the substrate with a minimal number of deposition steps, several parameters of the film buildup were optimized, such as the pH value of the solutions, the dipping time, and the salt (sodium chloride) concentration. The morphology of the films was characterized by atomic force microscopy. Built at an acidic pH, the stability of COL-based LbL films was studied when in contact with a physiological medium as well as the TA release from COL/TA films. In contrast to COL/PSS and COL/HEP LbL films, COL/TA films showed a good proliferation of human fibroblasts. These results validate the choice of TA and COL as components of LbL films for biomedical coatings.

Bioconjugation studies of an EGF-R targeting ligand on dendronized iron oxide nanoparticles to target head and neck cancer cells.

A major challenge in nanomedicine is designing nanoplatforms (NPFs) to selectively target abnormal cells to ensure early diagnosis and targeted therapy. Among developed NPFs, iron oxide nanoparticles (IONPs) are good MRI contrast agents and can be used for therapy by hyperthermia and as radio-sensitizing agents. Active targeting is a promising method for selective IONPs accumulation in cancer tissues and is generally performed by using targeting ligands (TL). Here, a TL specific for the epidermal growth factor receptor (EGFR) is bound to the surface of dendronized IONPs to produce nanostructures able to specifically recognize EGFR-positive FaDu and 93-Vu head and neck cancer cell lines. Several parameters were optimized to ensure a high coupling yield and to adequately quantify the amount of TL per nanoparticle. Nanostructures with variable amounts of TL on the surface were produced and evaluated for their potential to specifically target and be thereafter internalized by cells. Compared to the bare NPs, the presence of the TL at the surface was shown to be effective to enhance their internalization and to play a role in the total amount of iron present per cell.

Brush-Induced Orientation of Collagen Fibers in Layer-by-Layer Nanofilms: A Simple Method for the Development of Human Muscle Fibers.

The engineering of skeletal muscle tissue, a highly organized structure of myotubes, is promising for the treatment of muscle injuries and muscle diseases, for replacement, or for pharmacology research. Muscle tissue development involves differentiation of myoblasts into myotubes with parallel orientation, to ultimately form aligned myofibers, which is challenging to achieve on flat surfaces. In this work, we designed hydrogen-bonded tannic acid/collagen layer-by-layer (TA/COL LbL) nanofilms using a simple brushing method to address this issue. In comparison to films obtained by dipping, brushed TA/COL films showed oriented COL fibers of 60 nm diameter along the brushing direction. Built at acidic pH due to COL solubility, TA/COL films released TA in physiological conditions with a minor loss of thickness. After characterization of COL fibers' orientation, human myoblasts (C25CL48) were seeded on the oriented TA/COL film, ended by COL. After 12 days in a differentiation medium without any other supplement, human myoblasts were able to align on brushed TA/COL films and to differentiate into long aligned myotubes (from hundreds of µm up to 1.7 mm length) thanks to two distinct properties: (i) the orientation of COL fibers guiding myoblasts' alignment and (ii) the TA release favoring the differentiation. This simple and potent brushing process allows the development of anisotropic tissues in vitro which can be used for studies of drug discovery and screening or the replacement of damaged tissue.

Development of a biomimetic bioreactor for regenerative endodontics research.

In the context of regenerative endodontics research with the development of biomaterials, this work aimed to develop and test a prototype biomimetic bioreactor of a human tooth. The bioreactor was designed to reproduce a shaped dental canal connected with a cavity reproducing the periapical region and irrigated through two fluidic channels intended to reproduce the apical residual vascular supply. A test biomaterial composed of polylactic acid/polycaprolactone-tannic acid (PLA/PCL-TA) was produced by electrospinning/electrospraying and calibrated to be inserted in a dental canal. This biomaterial was first used to evaluate its imbibition capacity and the oximetry inside the bioreactor. Then, Dental Pulp Stem Cells (DPSCs) were cultured on PLA/PCL-TA cones for 1-3 weeks in the bioreactor; afterward cell adhesion, proliferation, and migration were histologically assessed. Complete imbibition biomaterial was obtained in 10 min and oximetry was stable over time. In the bioreactor, DPSCs were able to adhere, proliferate and migrate onto the surface and inside the biomaterial. In conclusion, this bioreactor was used successfully to test a biomaterial intended to support pulp regeneration and constitutes a new in vitro experimental model closer to clinical reality.

Impacts of Resveratrol and Pyrogallol on Physicochemical, Mechanical and Biological Properties of Epoxy-Resin Sealers.

This study aimed at evaluating the physicochemical and biological properties of experimental epoxy-resin sealers containing polyphenols such as resveratrol and pyrogallol. A conventional epoxy resin (OB) was modified by adding different concentrations of resveratrol (RS) or pyrogallol (PY) to its composition. Antibacterial and antioxidant activities, mechanical properties, along with wettability and morphological changes were investigated. The results were statistically analyzed using ANOVA and multiple comparison tests (α = 0.05). The incorporation of the tested polyphenols into the epoxy resin enhanced its mechanical properties. PY demonstrated much better antioxidant and antibacterial activities than RS, which were associated with a higher release of PY. In contrast, PY showed a higher cytotoxicity than OB and OB doped with RS. OB containing PY presented a rougher surface and higher water absorption than OB doped with RS. Both tested polyphenols caused no notable changes to the overall porosity of OB. Resveratrol and pyrogallol may not only influence the morphology and mechanical properties of epoxy-resin sealers, but could also enhance antioxidant activity and antibacterial effects against Enterococcus faecalis. Most epoxy-resin sealers currently available in the market can be considered as "passive" materials. Thus, doping their composition with specific polyphenols may be a suitable strategy to confer some antibacterial properties, antioxidant potential, along with improvement of some mechanical properties.

Development of an innovative educational escape game to promote teamwork in dentistry.

BACKGROUND: Escape games have proven to be an innovative pedagogical tool that allows students to use the professional skills they acquired. The appeal of the game lies in the stimulation of the players' minds and in the diversity of the puzzles. PURPOSE/OBJECTIVES: To evaluate the effectiveness of an educational tool aimed at cultivating team spirit and group cohesion in dentistry students through a fun collaborative activity that mobilises their knowledge and skills. MATERIALS AND METHOD: Twenty-four students participated to the escape game over a one-day period. In order to win, they had to solve dentistry-related puzzles. RESULTS: Feedback was strongly positive. The balance between manipulations and theoretical questions stimulated them. Students did favour this type of activity which allows to increase interactions between students as well as with the teaching team. CONCLUSION: Escape games in dental schools foster a supportive learning environment and stimulated students' motivation and group cohesion.

Physicochemical and Mechanical Properties of Premixed Calcium Silicate and Resin Sealers.

The aim of the present in vitro study was to evaluate specific mechanical and physicochemical properties of two calcium silicate based sealers, (AH Plus Bioceramic "AHPB"; Well-Root ST "WRST"), and a conventional resin sealer (AH Plus "AHP"). These aims were accomplished by assessing the porosity, pH, compression strength, roughness, wettability and cell attachment of the tested materials. The results were compared statistically using the one-way ANOVA test. Higher pH values were obtained in both AHPB and WRST compared to AHP at 3, 24 and 72 h (p < 0.05). A greater level of porosity and wettability was detected for both AHPB and WRST compared to the resin sealer AHP (p < 0.05). Evident cell growth characterized by elongated morphology was observed on the surface of AHPB and WRST, while only a thin layer of cells was seen on the surface of AHP. A significant lower compression strength and modulus were obtained in the specimens created using AHPB compared to those made with AHP and WRST (p < 0.05). The removal of calcium silicates may be quite tricky during endodontic retreatment. In conclusion, considering the limitations of the present in vitro study, both calcium silicate sealers demonstrated good physicochemical properties. However, the lower compression strength and modulus of AHPB may facilitate its removal and make the retreatment procedures considerably easier.

Highly Structured 3D Electrospun Conical Scaffold: A Tool for Dental Pulp Regeneration.

New procedures envisioned for dental pulp regeneration after pulpectomy include cell homing strategy. It involves host endogenous stem cell recruitment and activation. To meet this cell-free approach, we need to design a relevant scaffold to support cell migration from tissues surrounding the dental root canal. A composite membrane made of electrospun poly(lactic acid) nanofibers and electrosprayed polycaprolactone with tannic acid (TA) microparticles which mimics the architecture of the extracellular matrix was first fabricated. After rolling the membrane in the form of a 3D conical scaffold and subsequently coating it with gelatin, it can be directly inserted into the root canal. The porous morphology of the construct was characterized by SEM at different length scales. It was shown that TA was released from the 3D conical scaffold after 2 days in PBS at 37 °C. Biocompatibility studies were first assessed by seeding human dental pulp stem cells (DPSCs) on planar membranes coated or not coated with gelatin to compare the surfaces. After 24 h, the results highlighted that the gelatin-coating increased the membrane biocompatibility and cell viability. Similar DPSC morphology and proliferation on both membrane surfaces were observed. The culture of DPSCs on conical scaffolds showed cell colonization in the whole cone volume, proving that the architecture of the conical scaffold was suitable for cell migration.

Unveiling the role of surface, size, shape and defects of iron oxide nanoparticles for theranostic applications.

Iron oxide nanoparticles (IONPs) are well-known contrast agents for MRI for a wide range of sizes and shapes. Their use as theranostic agents requires a better understanding of their magnetic hyperthermia properties and also the design of a biocompatible coating ensuring their stealth and a good biodistribution to allow targeting of specific diseases. Here, biocompatible IONPs of two different shapes (spherical and octopod) were designed and tested in vitro and in vivo to evaluate their abilities as high-end theranostic agents. IONPs featured a dendron coating that was shown to provide anti-fouling properties and a small hydrodynamic size favoring an in vivo circulation of the dendronized IONPs. While dendronized nanospheres of about 22 nm size revealed good combined theranostic properties (r2 = 303 mM s-1, SAR = 395 W gFe-1), octopods with a mean size of 18 nm displayed unprecedented characteristics to simultaneously act as MRI contrast agents and magnetic hyperthermia agents (r2 = 405 mM s-1, SAR = 950 W gFe-1). The extensive structural and magnetic characterization of the two dendronized IONPs reveals clear shape, surface and defect effects explaining their high performance. The octopods seem to induce unusual surface effects evidenced by different characterization techniques while the nanospheres show high internal defects favoring Néel relaxation for magnetic hyperthermia. The study of octopods with different sizes showed that Néel relaxation dominates at sizes below 20 nm while the Brownian one occurs at higher sizes. In vitro experiments demonstrated that the magnetic heating capability of octopods occurs especially at low frequencies. The coupling of a small amount of glucose on dendronized octopods succeeded in internalizing them and showing an effect of MH on tumor growth. All measurements evidenced a particular signature of octopods, which is attributed to higher anisotropy, surface effects and/or magnetic field inhomogeneity induced by tips. This approach aiming at an analysis of the structure-property relationships is important to design efficient theranostic nanoparticles.

Dual role of tannic acid and pyrogallol incorporated in plaster of Paris: Morphology modification and release for antimicrobial properties.

The design of bioactive plasters is of major interest for the amelioration of dental and bone cements. In this article, a one pot and environmentally friendly strategy based on the addition of a cheap polyphenol-tannic acid (TA) or the main phenolic constituent of TA, namely pyrogallol (PY)- able to interact with calcium sulfate is proposed. Tannic acid and pyrogallol not only modify the morphology of the obtained plaster+TA/PY composites but a part of it is released and provides strong-up to twenty fold- antibacterial effect against Staphylococcus aureus. It is shown that the higher antibacterial efficiency of PY is related to a greater release compared to TA even if in solution the antibacterial effect of PY is lower than that of TA when reported on the basis of the molar concentration in PY units.

Microguided endodontics: Accuracy evaluation for access through intraroot fibre-post.

In this work, we intended to assess the reliability of guided endodontic technique to remove a bonded fibre-post when there are artefacts in the cone-beam computed tomography (CBCT) images caused by composite dental materials. We mounted natural posterior teeth on ten simulated models. Forty fibre-post and composite-core restorations were inserted in the teeth. We merged a pre-operative CBCT and optical surface scan on the BlueskyplanTM software to digitally design and subsequently 3D-printed the guides. Two operators initiated endodontic access into the fibre-post restorations using the template to guide the drill. Post-operative CBCT was taken and merged onto the pre-operative plan to measure the deviations at the coronal and apical segments. The mean deviation between the planned and actual drill paths were, respectively, of 0.39 ± 0.14 mm coronally and 0.40 ± 0.19 mm apically. Microguided endodontics is a predictable and accurate method to remove fibre-post restorations efficiently.

Effect of the Buffer on the Buildup and Stability of Tannic Acid/Collagen Multilayer Films Applied as Antibacterial Coatings.

The deposition of polyelectrolyte multilayers, obtained by the layer-by-layer (LbL) method, is a well-established technology to design biocompatible and antibacterial coatings aimed at preventing implant-associated infections. Several types of LbL films have been reported to exhibit antiadhesive and/or antibacterial (contact-killing or release-killing) properties governed not only by the incorporated compounds but also by their buildup conditions or their postbuildup treatments. Tannic acid (TA), a natural polyphenol, is known to inhibit the growth of several bacterial strains. In this work, we developed TA/collagen (TA/COL) LbL films built in acetate or citrate buffers at pH 4. Surprisingly, the used buffer impacts not only the physicochemical but also the antibacterial properties of the films. When incubated in physiological conditions, both types of TA/COL films released almost the same amount of TA depending on the last layer and showed an antibacterial effect against Staphylococcus aureus only for citrate-built films. Because of their granular topography, TA/COL citrate films exhibited an efficient release-killing effect with no cytotoxicity toward human gingival fibroblasts. Emphasis is put on a comprehensive evaluation of the physicochemical parameters driving the buildup and the antibacterial property of citrate films. Specifically, complexation strengths between TA and COL are different in the presence of the two buffers affecting the LbL deposition. This work constitutes an important step toward the use of polyphenols as an antibacterial agent when incorporated in LbL films.

Design of hybrid protein-coated magnetic core-mesoporous silica shell nanocomposites for MRI and drug release assessed in a 3D tumor cell model.

In this work, we describe the design and the use of a novel theranostic hybrid nanocomposite made of an iron oxide core and a mesoporous silica shell (IO@MS) of ca. 30 nm coated by human serum albumin (HSA) layer for magnetic resonance imaging and drug delivery applications. The porosity of IO@MS nanoparticles was loaded with an antitumoral drug, Doxorubicin (Dox) reaching a high drug loading capacity (DLC) of 34 w%. To entrap the drug, a tight HSA coating held via isobutyramide (IBAM) binders was deposited. We show that this protein nanoassembly entraps the drugs efficiently and behaves as an innovative enzyme-sensitive gatekeeper that is degraded upon protease action. Finally we assess the Dox release in a 3D cell model via confocal imaging and its cytotoxicity is shown by growth inhibition studies on liver cancer cell spheroids.

Mesoporous silica templated-albumin nanoparticles with high doxorubicin payload for drug delivery assessed with a 3-D tumor cell model.

Human serum albumin (HSA) nanoparticles emerge as promising carriers for drug delivery. Among challenges, one important issue is the design of HSA nanoparticles with a low mean size of ca. 50 nm and having a high drug payload. The original strategy developed here is to use sacrificial mesoporous nanosilica templates having a diameter close to 30 nm to drive the protein nanocapsule formation. This new approach ensures first an efficient high drug loading (ca. 30%) of Doxorubicin (DOX) in the porous silica by functionalizing silica with an aminosiloxane layer and then allows the one-step adsorption and the physical cross-linking of HSA by modifying the silica surface with isobutyramide (IBAM) groups. After silica template removal, homogenous DOX-loaded HSA nanocapsules (30-60 nm size) with high drug loading capacity (ca. 88%) are thus formed. Such nanocapsules are shown efficient in multicellular tumor spheroid models (MCTS) of human hepatocarcinoma cells by their significant growth inhibition with respect to controls. Such a new synthesis approach paves the way toward new protein based nanocarriers for drug delivery.

Mimicking the Chemistry of Natural Eumelanin Synthesis: The KE Sequence in Polypeptides and in Proteins Allows for a Specific Control of Nanosized Functional Polydopamine Formation.

The oxidation of dopamine and of other catecholamines leads to the formation of conformal films on the surface of all known materials and to the formation of a precipitate in solution. In some cases, it has been shown that the addition of additives in the dopamine solution, like certain surfactants or polymers, polyelectrolytes, and certain proteins, allows to get polydopamine nanoparticles of controlled size and the concomitant decrease, in an additive/dopamine dependent manner, in film formation on the surface of the reaction beaker. However, the mechanism behind this controlled oxidation and self-assembly of catecholamines is not known. In this article, it is shown that a specific diad of amino acids in proteins, namely KE, allows for specific control in the oxidation-self-assembly of dopamine to obtain polydopamine@protein core-shell nanoparticles which are biocompatible. The interactions between dopamine and the adjacent KE amino acids potentially responsible for the size control of polydopamine aggregates was investigated by molecular dynamics simulations. The obtained core-shell nanoparticles display the biological activity of the protein used to control the self-assembly of PDA. The photon to heat conversion ability of PDA is conserved in the PDA@protein particles.

Polyphenols at interfaces.

Polyphenols are important molecules in living organisms, particularly in plants, where they serve as protectants against predators. They are also of fundamental importance in pharmacology for their antioxidant and antibacterial activities. Since a few years polyphenols are also used in surface functionalization mimicking the tannin deposition observed when tea or red wine are in contact with the surface of cups or glasses respectively. The interaction of polyphenols with proteins to yield colloids and of polyphenol with surfaces will be reviewed in this article to provide an overview of such particles and surface functionalization methods in modern surface science. Particular emphasis will be given to biological applications of polyphenols at interfaces.

Polymer-free electrospinning of tannic acid and cross-linking in water for hybrid supramolecular nanofibres.

Electrospinning is the process of choice allowing the preparation of nanofibrous materials from a solution usually based on a high molar mass polymer. The solution must bring enough chain entanglements to avoid any breaking or Rayleigh instability of the electrospun jet resulting thus in the deposition of a continuous and regular solid nanofibre. It has been however shown that some few non-polymeric molecules can be electrospun without using a carrier polymer. We demonstrate here the case of tannic acid. Indeed, it was possible to electrospin this molecule solubilised in a mixture of water and ethanol as well as in pure water. Rheology, dynamic light scattering and cryo-TEM highlight the formation of tannic acid aggregates in solution. Above a critical concentration, these aggregates form a supramolecular interconnected network strong enough to allow the electrospinning of a continuous and regular nanofibre. The resulting nanoweb is mechanically stable and can be handled and wrapped. Furthermore, as opposed to the other small molecules for which polymer-free electrospinning was also demonstrated, tannic acid nanowebs can be efficiently cross-linked in water either by oxidative reaction with sodium periodate or, most interestingly, with FeIII by a combination of oxidative reaction and the formation of coordination complexes. The proposed electrospinning and cross-linking strategy is easy, of low cost, and scalable and uses non-toxic solvents as well as biocompatible and biofunctional molecules. Furthermore, thanks to the chelation capacity of tannic acid having the ability to coordinate with a wide variety of metals, hybrid smart nanowebs can be envisaged for diverse applications such as biomedical, catalysis as well as environment.

Multilayered films made from tannic acid and alkaline phosphatasewith enzymatic activity and electrochemical behavior.

Layer-by-layer-deposition of enzymes and polyphenols, like tannic acid may provide a reservoir of antioxidant and antibacterial molecules of controlled thickness and degradability with an additional activity due to the presence of the enzyme. The layer-by-layer deposition of films made from tannic acid and alkaline phosphatase is shown to yield an exponential growth with the number of deposited layer pairs. The films display the electrochemical behavior of tannic acid and the enzymatic activity of alkaline phosphatase. However, it is shown that only the enzyme close to the film-solution interface is active and follows the Michaelis-Menten mechanism. Similarly, only tannic acid close to the electrode-film interface can be oxidized. The enzymatic activity is almost completely lost when the multilayer film is treated with sodium periodate which oxidizes tannic acid even if the solubilized enzyme is not affected by the oxidant. This shows that the formation of covalent bonds between alkaline phosphatase and tannic acid is deleterious for its conformation and activity.

Performance of Pyridylthiourea-Polyethylenimine Polyplex for siRNA-Mediated Liver Cancer Therapy in Cell Monolayer, Spheroid, and Tumor Xenograft Models.

Medical application of siRNAs relies on methods for delivering nucleic acids into the cytosol. Synthetic carriers, which assemble with nucleic acids into delivery systems, show promises for cancer therapy but efficiency remains to be improved. In here, the effectiveness of pyridylthiourea-polyethylenimine (πPEI), a siRNA carrier that favors both polyplex disassembly and endosome rupture upon sensing the acidic endosomal environment, in 3 experimental models of hepatocellular cancer is tested. The πPEI-assisted delivery of a siRNA targeting the polo-like kinase 1 into Huh-7 monolayer produces a 90% cell death via a demonstrated RNA interference mechanism. Incubation of polyplex with Huh-7 spheroids leads to siRNA delivery into the superficial first cell layer and a 60% reduction in spheroid growth compared to untreated controls. Administration of polyplexes into mice bearing subcutaneous implanted Huh-7Luc tumors results in a reduced tumor progression, similar to the one observed in the spheroid model. Altogether, these results support the in vivo use of synthetic and dedicated polymers for increasing siRNA-mediated gene knockdown, and their clinical promise in cancer therapeutics.