Curcumin Modulates 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) Liposomes: Chitosan Oligosaccharide Lactate Influences Membrane Fluidity But Does Not Alter Phase Transition Temperature of DBPC Liposomes.

1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) is one of the important phospholipids found in cell membrane but not studied well. Importance of curcumin as a dietary supplement and for its medicinal properites is getting widely recoginsed. The present study for the first time explores the effect of curcumin on properties of DBPC liposomes by monitoring the fluorescence properties of curcumin. The phase transition temperature (Tm) of DBPC is assessed which confirms increase in curcumin concentration causes a slight drop in the Tm value. Chitosan is being applied for various drug delivery uses. The study establishes new insight on effect of chitosan oligosaccharide lactate on DBPC liposomes. It is found that in the absence of chitosan oligosaccharide lactate, curcumin partitions more strongly in the liquids crystalline phase than in the solid gel phase, however, the opposite result is obtained with the presence of chitosan oligosaccharide lactate which penetrates into the DBPC liposomes membranes at higher temperature, blocking thus the passage of curcumin into the lipid bilayer. However, addition of chitosan oligosaccharide lactate had no effect on the Tm. Fluorescence quenching study of curcumin establishes that the location of curcumin to be in the hydrophobic cavity of DBPC membrane.

Preparation of External Stimulus-Free Gelatin-Catechol Hydrogels with Injectability and Tunable Temperature Responsiveness.

Gelatin is one of the most versatile biopolymers in various biomedical applications. A gelatin derivative gelatin-catechol (Gel-C) was developed in this study to further optimize its chemical and physical properties such as thermal reversibility and injectability. We found that Gel-C remains in a solution state at room temperature, and the temperature-dependent gelation capability of gelatin is well preserved in Gel-C. Its gel-forming temperature decreased to about 10 °C (about 30 °C for gelatin), and a series of gelatin derivatives with different gel-forming temperatures (10-30 °C) were formed by mixing gelatin and Gel-C in different ratios. Additionally, irreversible Gel-C hydrogels could be made without the addition of external stimuli by combining the physical cross-linking of gelatin and the chemical cross-linking of catechol. At the same time, properties of Gel-C hydrogels such as thermal reversibility and injectability could be manipulated by controlling the temperature and pH of the precursor solution. By simulating the formation of an irreversible Gel-C hydrogel in vivo, an in situ gelling system was fabricated by lowering the local temperature of the hydrogel with cold shock, thus realizing targeted and localized molecular delivery with prolonged retention time. This simple system integrated with the temperature responsiveness of gelatin and chemical cross-linking of catechol groups thus provides a promising platform to fabricate an in situ gelling system for drug delivery.

Structural Water Stabilizes Protein Motifs in Liquid Protein Phase: The Folding Mechanism of Short ß-Sheets Coupled to Phase Transition.

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small ß-sheets, the key motifs of proteins, undergoing structural changes in liquid-liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3-0.5 kcal·mol-1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2-2.3 kcal·mol-1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal·mol-1 per residue excess stabilization.

Is the Membrane Lipid Matrix a Key Target for Action of Pharmacologically Active Plant Saponins?

This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and triterpenes were tested. A systematic analysis of the effects of these chemicals on the physicochemical properties of the lipid bilayers and on the formation and functionality of the reconstituted ion channels induced by antimicrobial agents was performed. It was found that digitonin, tribulosin, and dioscin substantially reduced the boundary potential of the phosphatidylcholine membranes. We concluded that saponins might affect the membrane boundary potential by restructuring the membrane hydration layer. Moreover, an increase in the conductance and lifetime of gramicidin A channels in the presence of tribulosin was due to an alteration in the membrane dipole potential. Differential scanning microcalorimetry data indicated the key role of the sapogenin core structure (steroid or triterpenic) in affecting lipid melting and disordering. We showed that an alteration in pore forming activity of syringomycin E by dioscin might be due to amendments in the lipid packing. We also found that the ability of saponins to disengage the fluorescent marker calcein from lipid vesicles might be also determined by their ability to induce a positive curvature stress.

Intrinsically disordered proteins and biomolecular condensates as drug targets.

Intrinsically disordered domains represent attractive therapeutic targets because they play key roles in cancer, as well as in neurodegenerative and infectious diseases. They are, however, considered undruggable because they do not form stable binding pockets for small molecules and, therefore, have not been prioritized in drug discovery. Under physiological solution conditions many biomedically relevant intrinsically disordered proteins undergo phase separation processes leading to the formation of mesoscopic highly dynamic assemblies, generally known as biomolecular condensates that define environments that can be quite different from the solutions surrounding them. In what follows, we review key recent findings in this area and show how biomolecular condensation can offer opportunities for modulating the activities of intrinsically disordered targets.

Combined Effect of the Preparation Method and Compression on the Physical Stability and Dissolution Behavior of Melt-Quenched Amorphous Celecoxib.

In an earlier investigation, amorphous celecoxib was shown to be sensitive to compression-induced destabilization. This was established by evaluating the physical stability of uncompressed/compressed phases in the supercooled state (Be̅rzins . Mol. Pharmaceutics, 2019, 16(8), 3678-3686). In this study, we investigated the ramifications of compression-induced destabilization in the glassy state as well as the impact of compression on the dissolution behavior. Slow and fast melt-quenched celecoxib disks were compressed with a range of compression pressures (125-500 MPa) and dwell times (0-60 s). These were then monitored for crystallization using low-frequency Raman spectroscopy when kept under dry (∼20 °C; <5% RH) and humid (∼20 °C; 97% RH) storage conditions. Faster crystallization was observed from the samples, which were compressed using more severe compression parameters. Furthermore, crystallization was also affected by the cooling rate used to form the amorphous phases; slow melt-quenched samples exhibited higher sensitivity to compression-induced destabilization. The behavior of the melt-quench disks, subjected to different compression conditions, was continuously monitored during dissolution using low-frequency Raman and UV/vis for the solid-state form and dissolution properties, respectively. Surprisingly the compressed samples exhibited higher apparent dissolution (i.e., higher area under the dissolution curve and initial celecoxib concentration in solution) than the uncompressed samples; however, this is attributed to biaxial fracturing throughout the compressed compacts yielding a greater effective surface area. Differences between the slow and fast melt quenched samples showed some trends similar to those observed for their storage stability.

Impact of the Chain Length and Topology of the Acetylated Oligosaccharide on the Crystallization Tendency of Naproxen from Amorphous Binary Mixtures.

The impact of the chain length or dispersity of polymers in controlling the crystallization of amorphous active pharmaceutical ingredients (APIs) has been discussed for a long time. However, because of the weak control of these parameters in the majority of macromolecules used in pharmaceutical formulations, the abovementioned topic is poorly understood. Herein, four acetylated oligosaccharides, maltose (acMAL), raffinose (acRAF), stachyose (acSTA), and α-cyclodextrin (ac-α-CD) of growing chain lengths and different topologies (linear vs cyclic), mimicking the growing backbone of the polymer, were selected to probe the influence of these structural factors on the crystallization of naproxen (NAP)-an API that does not vitrify regardless of the cooling rate applied in our experiment. It was found that in equimolar systems composed of NAP and linear acetylated oligosaccharides, the progress and activation barrier for crystallization are dependent on the molecular weight of the excipient despite the fact that results of Fourier transform infrared studies indicated that there is no difference in the interaction pattern between measured samples. On the other hand, complementary dielectric, calorimetric, and X-ray diffraction data clearly demonstrated that NAP mixed with ac-α-CD (cyclic saccharide) does not tend to crystallize even in the system with a much higher content of APIs. To explain this interesting finding, we have carried out further density functional theory computations, which revealed that incorporation of NAP into the cavity of ac-α-CD is hardly possible because this state is of much higher energy (up to 80 kJ/mol) with respect to the one where the API is located outside of the saccharide torus. Hence, although at the moment, it is very difficult to explain the much stronger impact of the cyclic saccharide on the suppression of crystallization and enhanced stability of NAP with respect to the linear carbohydrates, our studies clearly showed that the chain length and the topology of the excipient play a significant role in controlling the crystallization of this API.

The prion-like domain of Fused in Sarcoma is phosphorylated by multiple kinases affecting liquid- and solid-phase transitions.

Fused in Sarcoma (FUS) is a ubiquitously expressed protein that can phase-separate from nucleoplasm and cytoplasm into distinct liquid-droplet structures. It is predominantly nuclear and most of its functions are related to RNA and DNA metabolism. Excessive persistence of FUS within cytoplasmic phase-separated assemblies is implicated in the diseases amyotrophic lateral sclerosis and frontotemporal dementia. Phosphorylation of FUS's prion-like domain (PrLD) by nuclear phosphatidylinositol 3-kinase-related kinase (PIKK)-family kinases following DNA damage was previously shown to alter FUS's liquid-phase and solid-phase transitions in cell models and in vitro. However, proteomic data suggest that FUS's PrLD is phosphorylated at numerous additional sites, and it is unknown if other non-PIKK and nonnuclear kinases might be influencing FUS's phase transitions. Here we evaluate disease mutations and stress conditions that increase FUS accumulation into cytoplasmic phase-separated structures. We observed that cytoplasmic liquid-phase structures contain FUS phosphorylated at novel sites, which occurred independent of PIKK-family kinases. We engineered phosphomimetic substitutions within FUS's PrLD and observed that mimicking a few phosphorylation sites strongly inhibited FUS solid-phase aggregation, while minimally altering liquid-phase condensation. These effects occurred independent of the exact location of the phosphomimetic substitutions, suggesting that modulation of PrLD phosphorylation may offer therapeutic strategies that are specific for solid-phase aggregation observed in disease.

Phase separation as a therapeutic target in tight junction-associated human diseases.

Tight junctions (TJs) play an important role in the maintenance of epithelial and endothelial barriers. Zonula occludens (ZO) proteins are scaffolding molecules essential for the formation of TJ complexes, and abnormalities in ZO proteins have been implicated in various TJ-associated human diseases such as tumor invasion and metastasis, and barrier dysfunction. Recent studies reveal that liquid-liquid phase separation of ZO proteins drives the polymerization of TJ proteins into a continuous belt, which then recruits various proteins to form the TJ complex to regulate selective paracellular permeability and signal transduction. Herein, we describe recent advances on how ZO phase separation contributes to TJ formation and discuss the potential of phase separation as a target for the treatment of TJ-associated diseases.

The effects of Levothyroxine on the structure and dynamics of DPPC liposome: FTIR and DSC studies.

Levothyroxine (3,5,3',5'-tetraiodothyronine), which is a L-isomer of thyroxine T4 (L-T4), is a synthetic thyroid hormone that is biochemically and physiologically indistinguishable from endogenous T4. It is used as a thyroid hormone replacement drug to treat an underactive thyroid gland. The interaction of L-T4, with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) was studied in the presence (1 mol%, 3 mol%, 6 mol%, 9 mol%, 15 mol%, 24 mol% and 30 mol%) and absence of L-T4 by using two different non-invasive techniques; Fourier Transform Infrared (FTIR) spectroscopy and Differential Scanning Calorimetry (DSC). The results show that L-T4 does perturb the phase transition profile by either decreasing the main transition temperature (Tm) and enthalpy (ΔH) or increasing the width at half height (ΔT½). That means; it changes the physical properties of DPPC bilayers. Addition of L-T4 into pure DPPC liposomes shifts the phase transition to lower temperature, disorder the system in gel phase with opposite effect in liquid-crystalline phase and increases the dynamics of the system in both phases and also causes dehydration of the groups of lipids and the water molecules around.

Stimuli-sensitive cross-linked hydrogels as drug delivery systems: Impact of the drug on the responsiveness.

Responsiveness of drug delivery systems (DDS) against internal and external stimuli attracts wide interest as a mechanism that can provide both site-specific release at the target place and feedback regulated release rate. Biological environment is quite complex and the effects that the intricate medium may have on the effectiveness of the stimulus have received certain attention. Differently, the impact that the drug loaded may have itself on the responsiveness of the DDS has been underestimated. Most drugs are not merely trapped in the polymer network, but they effectively interact with some polymer moieties. Nearly all drugs, including therapeutic proteins, are ionizable amphiphilic molecules, and thus ionic, hydrogen bonding and hydrophobic interactions are commonly exploited to increase the loading yield. If the moiety involved in drug binding is also responsible for (or at least partially involved in) the stimuli responsiveness, a strong impact of the drug on the behavior of the DDS can be expected. This review gathers relevant examples of how the drug may modify the sensitiveness (stimulus threshold) and the responsiveness (actuation) of the DDS to therapeutically relevant stimulus, and aims to shed light on the different drug binding modes of the swollen and collapsed states, which in turn modify drug release patterns. The information evidences that drug loading and release may trigger phase transitions in hydrogels non-intended to be drug-responsive (i.e., a priori not analyte-responsive networks). A better knowledge about the effect of the drug on the responsiveness is a required step forward for the clinical application of smart hydrogels and may also unveil novel uses of the stimuli-responsive DDS.

Investigation of drug-polymer miscibility, biorelevant dissolution, and bioavailability improvement of Dolutegravir-polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer solid dispersions.

The aim of the current study was to prepare the efficacious amorphous solid dispersion of poorly water-soluble compound, Dolutegravir. After theoretical and experimental determination of drug-polymer miscibility, polyvinyl caprolactam-polyvinyl acetate-polyethylne glycol graft copolymer was chosen as a polymer. The solid dispersions of Dolutegravir were prepared by quench cooling and solvent evaporation method. Though quench cooling successfully stabilized the drug into amorphous form, solvent evaporation technique failed to render the drug completely amorphous. Owing to the negative Gibbs free energy at room temperature, the prepared dispersions were found stable at room temperature for 60 days. To resolve the overlapping contribution of micellar solubilization and amorphicity in improving the dissolution characteristics of Dolutegravir, the in vitro dissolution studies were performed in USP phosphate buffer as well as bio-relevant media. The dissolution advantage between prepared dispersions and pure drug in USP phosphate buffer was found bridged in the bio-relevant media. For this, the micellar solubilization driven dissolution of Dolutegravir in the presence of bile and lecithin micelles was thought as a contributing factor. Nevertheless, the dissolution advantage of dispersions prepared by quench cooling method was found endured in FeSSIF, which was thought to be due to its amorphicity leading to molecular level dissolution. Subsequently, the dissolution advantage was translated into the improved flux. Further, in vivo oral bioavailability was investigated for the dispersion prepared by quench cooling by using crystalline Dolutegravir as a control. The overall exposure of Dolutegravir was improved by 1.7 fold (AUC), while the maximum plasma concentration (Cmax) demonstrated 2 fold increase after comparing with crystalline Dolutegravir.

Solvation properties of raft-like model membranes.

Dimethyl sulfoxide (DMSO) is a universal water-soluble solvent widely used in many biotechnological and medical applications, such as cells cryopreservation, and for the treatment of different human diseases (e.g. amyloidosis). Despite the great number of reported studies, the effects of DMSO on the physico-chemical properties of biological membranes are poorly understood. Often, these studies are limited to model membranes composed of phosphatidylcholines (PCs) and cholesterol (Chol). In this work, we explored the effect of DMSO on liposomes composed of the natural egg sphingomyelin (ESM) and Chol as raft-like model membranes. With a multi-technique approach we probe the structure and the thermal stability of ESM/Chol bilayer at different Chol mole fractions. In particular, we investigate the ESM-solvent interactions to clarify the role of DMSO in perturbing the solvating conditions of lipid vesicles and show that the addition of DMSO increases the thermal stability of vesicles. An increase of transition temperature, a decrease of both enthalpy and entropy as well as a decrease of the cooperativity of the gel to liquid phase transition are observed at 0.1 DMSO mole fraction. Fluorescence experiments with the probe Laurdan and FTIR spectra strongly indicate that DMSO exerts a dehydration effect on the membrane. Besides, FTIR measurements with tungsten hexacarbonyl, in combination with fluorescence data of the probe NBD-PE, indicate that DMSO promotes the formation of a highly packed membrane by reducing the thickness of the membrane.

CETSA beyond Soluble Targets: a Broad Application to Multipass Transmembrane Proteins.

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.

Temperature- and rigidity-mediated rapid transport of lipid nanovesicles in hydrogels.

Lipid nanovesicles are widely present as transport vehicles in living organisms and can serve as efficient drug delivery vectors. It is known that the size and surface charge of nanovesicles can affect their diffusion behaviors in biological hydrogels such as mucus. However, how temperature effects, including those of both ambient temperature and phase transition temperature (Tm), influence vehicle transport across various biological barriers outside and inside the cell remains unclear. Here, we utilize a series of liposomes with different Tm as typical models of nanovesicles to examine their diffusion behavior in vitro in biological hydrogels. We observe that the liposomes gain optimal diffusivity when their Tm is around the ambient temperature, which signals a drastic change in the nanovesicle rigidity, and that liposomes with Tm around body temperature (i.e., ∼37 °C) exhibit enhanced cellular uptake in mucus-secreting epithelium and show significant improvement in oral insulin delivery efficacy in diabetic rats compared with those with higher or lower Tm Molecular-dynamics (MD) simulations and superresolution microscopy reveal a temperature- and rigidity-mediated rapid transport mechanism in which the liposomes frequently deform into an ellipsoidal shape near the phase transition temperature during diffusion in biological hydrogels. These findings enhance our understanding of the effect of temperature and rigidity on extracellular and intracellular functions of nanovesicles such as endosomes, exosomes, and argosomes, and suggest that matching Tm to ambient temperature could be a feasible way to design highly efficient nanovesicle-based drug delivery vectors.

Influence of a hydrophobic monomer on the physical and mechanical properties of experimental surface sealants.

This study evaluated the effect of adding the hydrophobic monomer 1,12 dodecanediol dimethacrylate (DDDMA) to experimental sealants with and without thermocycling on degree of conversion (DC), water sorption (WS), water solubility (WSB), color stability (ΔE), and micro-shear bond strength (µSBS). Five experimental and one commercially available sealant (Bisco - BIS) were tested. The experimental sealants were formulated by mixing different percentages of DDDMA monomers and urethane dimethacrylate (UDMA). The photoinitiator system was composed by camphorquinone (CQ) and tertiary amine 4-ethyl benzoate dimetilamiono (EDBA). Ethanol was used as a solvent. The experimental groups were named sequentially according to the monomeric content (DDDMA/UDMA): S40/40 (40/40), S50/30 (50/30), S60/20 (60/20), S70/10 (70/10) and S80/0 (80/0). Data were analyzed separately by one-way ANOVA, followed by Tukey's test (p<0.05). The values of DC ranged from 94.59% (S40/40) to 54.02% (S80/10). BIS showed the highest WS value (p<0.05) and S40/40, S50/30, S60/20 and S80/0 showed the lowest WS values of all tested sealants. WSB values ranged from 7.88 µg/mm3 (BIS) to 13.27 µg/mm3 (S70/10). The highest ΔE value was 11.05±2.88 for BIS and the highest µSBS value was found for S60/20. No significant difference was observed in bond strength between sealants and bovine enamel after thermocycling. Adding DDDMA to the composition of surface sealants can improve its performance, once the monomer increased the degree of conversion and the color stability.

Molecular nucleation mechanisms and control strategies for crystal polymorph selection.

The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts, amyotrophic lateral sclerosis, sickle cell anaemia and Alzheimer's disease. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures, or are used as delivery vehicles for pharmaceutical applications. The physiochemical properties of crystals can vary substantially between different forms or structures ('polymorphs') of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein-based drug-delivery systems and macromolecular crystallography.

Cardiomyocyte-targeted and 17ß-estradiol-loaded acoustic nanoprobes as a theranostic platform for cardiac hypertrophy.

BACKGROUND: Theranostic perfluorocarbon nanoprobes have recently attracted attention due to their fascinating versatility in integrating diagnostics and therapeutics into a single system. Furthermore, although 17ß-estradiol (E2) is a potential anti-hypertrophic drug, it has severe non-specific adverse effects in various organs. Therefore, we have developed cardiomyocyte-targeted theranostic nanoprobes to achieve concurrent targeted imaging and treatment of cardiac hypertrophy. RESULTS: We had successfully synthesized E2-loaded, primary cardiomyocyte (PCM) specific peptide-conjugated nanoprobes with perfluorocarbon (PFP) as a core (PCM-E2/PFPs) and demonstrated their stability and homogeneity. In vitro and in vivo studies confirmed that when exposed to low-intensity focused ultrasound (LIFU), these versatile PCM-E2/PFPs can be used as an amplifiable imaging contrast agent. Furthermore, the significantly accelerated release of E2 enhanced the therapeutic efficacy of the drug and prevented systemic side effects. PCM-E2/PFPs + LIFU treatment also significantly increased cardiac targeting and circulation time. Further therapeutic evaluations showed that PCM-E2/PFPs + LIFU suppressed cardiac hypertrophy to a greater extent compared to other treatments, revealing high efficiency in cardiac-targeted delivery and effective cardioprotection. CONCLUSION: Our novel theranostic nanoplatform could serve as a potential theranostic vector for cardiac diseases.

Evaluation of long-term bond strength and selected properties of self-adhesive resin cements.

This study evaluated the shear bond strength (SBS) of self-adhesive resin cements (SARCs) to dentin and their physical-chemical properties. Five commercial SARCs were evaluated [SmartCem®2 - DENTSPLY (SC2); BisCem® - Bisco (BC); SeT PP® - SDI (SeT); Relyx U100® - 3M ESPE (U100) and YCEM® SA - Yller (YCEM)]. The SARCs were evaluated for SBS to dentin (n = 10) after 24 h, 6 months, and 12 months. The dentin demineralization caused by acidic monomers was observed by SEM, and pH-neutralization of eluate was observed for 24 h. Degree of conversion (DC), rate of polymerization (Rp), flexural strength (FS), and elastic modulus (E) were evaluated. Immediate SBS of SC2, SET, U100, and YCEM were statistically higher than that of BC (p < 0.001). After 12 months, all SARCs showed reduced SBS values and U100 showed values similar to those of SET and YCEM, and higher than those of BC and SC2 (p = 0.001). Demineralization pattern of SARCs was similar. At 24h, all SARCs showed no differences in the pH-value, except BC and U100 (p < 0.001). YCEM showed the highest Rp. U100, YCEM, and SC2 showed statistically higher FS (p<0.001) and E (p < 0.001) when compared with SET and BC. U100 and YCEM showed the best long-term bonding irrespective of the storage period. A significant reduction in SBS was found for all groups after 12 months. SBS was not shown to be correlated with physical-chemical properties, and appeared to be material-dependent. The polymerization profile suggested that an increased time of light activation, longer than that recommended by manufacturers, would be necessary to optimize DC of SARCs.

Effect on adhesion of a nanocapsules-loaded adhesive system.

This study aimed to evaluate the in situ degree of conversion, contact angle, and immediate and long-term bond strengths of a commercial primer and an experimental adhesive containing indomethacin- and triclosan-loaded nanocapsules (NCs). The indomethacin- and triclosan-loaded NCs, which promote anti-inflammatory and antibacterial effects through controlled release, were incorporated into the primer at a concentration of 2% and in the adhesive at concentrations of 1, 2, 5, and 10%. The in situ degree of conversion (DC, n=3) was evaluated by micro-Raman spectroscopy. The contact angle of the primer and adhesive on the dentin surface (n = 3) was determined by an optical tensiometer. For the microtensile bond strength µTBS test (12 teeth per group), stick-shaped specimens were tested under tensile stress immediately after preparation and after storage in water for 1 year. The data were analyzed using two-way ANOVA, three-way ANOVA and Tukey's post hoc tests with α=0.05. The use of the NC-loaded adhesive resulted in a higher in situ degree of conversion. The DC values varied from 75.07 ± 8.83% to 96.18 ± 0.87%. The use of NCs in only the adhesive up to a concentration of 5% had no influence on the bond strength. The contact angle of the primer remained the same with and without NCs. The use of both the primer and adhesive with NCs (for all concentrations) resulted in a higher contact angle of the adhesive. The longitudinal µTBS was inversely proportional to the concentration of NCs in the adhesive system, exhibiting decreasing values for the groups with primer containing NCs and adhesives with increasing concentrations of NCs. Adhesives containing up to 5% of nanocapsules and primer with no NCs maintained the in situ degree of conversion, contact angle, and immediate and long-term bond strengths. Therefore, the NC-loaded adhesive can be an alternative method for combining the bond performance and therapeutic effects. The use of an adhesive with up to 5% nanocapsules containing indomethacin and triclosan and a primer with no nanocapsules maintained the long-term bond performance.