Effects of deep heating modalities on the morphological and elastic properties of the non-insertional region of achilles tendon: a pilot study.

BACKGROUND: Over the last 20 years, both diathermy and ultrasound have been popular choices for many clinicians in treating musculoskeletal disorders. However, there is a lack of clinical evidence of deep heating modalities to treat tendon pathology, There is no study to investigate the effects of such as physical modalities on morphological and elastic properties on the human tendons. OBJECTIVE: the objective of the present study was to compare the effects of diathermy and ultrasound therapies on cross sectional area, transversal height and hardness percentage of the non-insertional region of the Achilles tendon in able-bodied subjects. METHODS: healthy volunteers were divided in diathermy and ultrasound group received six 15-min treatment sessions. Before and after treatment a sonographic assessment was conducted by mean of ultrasonography and the following parameters were recorded: cross sectional area, transversal height and hardness percentage. RESULTS: thirty-two subjects were enrolled. Between-group comparisons showed a significant change on hardness percentage (p = 0.004) after treatment in diathermy therapy group. Within-group comparison showed a significant improvement in the hardness percentage for the diathermy (p = 0.001) and ultrasound (p = 0.046) after two weeks of treatment. CONCLUSION: this pilot study demonstrated larger effects on morphological and elastic properties of the non-insertional region of the Achilles tendon after diathermy than ultrasound therapy in normal tendons. Diathermy may be a useful deep heat modality for treating non-insertional Achilles tendinopathy.

Unprecedented Behavior of (9 R)-9-Hydroxystearic Acid-Loaded Keratin Nanoparticles on Cancer Cell Cycle.

Histone deacetylases, HDACs, have been demonstrated to play a critical role in epigenetic signaling and were found to be overexpressed in several type of cancers; therefore, they represent valuable targets for anticancer therapy. 9-Hydroxystearic acid has been shown to bind the catalytic site of HDAC1, inducing G0/G1 phase cell cycle arrest and activation of the p21WAF1 gene, thus promoting cell growth inhibition and differentiation in many cancer cells. Despite the ( R) enantiomer of 9-hydroxystearic acid (9R) displaying a promising in vitro growth-inhibitory effect on the HT29 cell line, its scarce water solubility and micromolar activity require novel solutions for improving its efficacy and bioavailability. In this work, we describe the synthesis and in vitro biological profiling of 9R keratin nanoparticles (9R@ker) obtained through an in-water drug-induced aggregation process. The anticancer activity of 9R@ker was investigated in the HT29 cell line; the results indicate an increased fluidity of cell membrane and a higher intracellular ROS formation, resulting in an unexpected S phase cell cycle arrest (25% increase as compared to the control) induced by 9R@ker with respect to free 9R and an induction of cell death.

Functionalized Keratin as Nanotechnology-Based Drug Delivery System for the Pharmacological Treatment of Osteosarcoma.

Osteosarcoma therapy might be moving toward nanotechnology-based drug delivery systems to reduce the cytotoxicity of antineoplastic drugs and improve their pharmacokinetics. In this paper, we present, for the first time, an extensive chemical and in vitro characterization of dual-loaded photo- and chemo-active keratin nanoparticles as a novel drug delivery system to treat osteosarcoma. The nanoparticles are prepared from high molecular weight and hydrosoluble keratin, suitably functionalized with the photosensitizer Chlorin-e6 (Ce6) and then loaded with the chemotherapeutic drug Paclitaxel (PTX). This multi-modal PTX-Ce6@Ker nanoformulation is prepared by both drug-induced aggregation and desolvation methods, and a comprehensive physicochemical characterization is performed. PTX-Ce6@Ker efficacy is tested on osteosarcoma tumor cell lines, including chemo-resistant cells, using 2D and 3D model systems. The single and combined contributions of PTX and Ce6 is evaluated, and results show that PTX retains its activity while being vehiculated through keratin. Moreover, PTX and Ce6 act in an additive manner, demonstrating that the combination of the cytostatic blockage of PTX and the oxidative damage of ROS upon light irradiation have a far superior effect compared to singularly administered PTX or Ce6. Our findings provide the proof of principle for the development of a novel, nanotechnology-based drug delivery system for the treatment of osteosarcoma.

Levers supporting tariff growth for water services: evidence from a contingent valuation analysis.

The backwardness of the water utilities sector necessitates urgent investment in infrastructure to improve water quality and efficiency in water supply networks. A policy of tariff growth represents the main source to sustain such investments. Therefore, customer engagement in the form of willingness to pay (WTP) is highly desirable by water utilities to obtain social legitimization and support. This study examines the determinants of consumers' WTP for improvement programs for three drinking water issues: quality of water sources, renewal of water mains, and building of new wastewater treatment plants. The study is based on a survey conducted among a sample of 587 customers of a water utility located in the province of Verona in the north of Italy. The contingence valuation method is used to measure WTP. Specifically, an ordinal logistic regression model yields the following significant determinants of WTP: quality of water and services provided, preference for privatization of the water utility, sustainable consumption of water, and some socio-demographic variables. The findings provide interesting insights into the drivers of WTP as well as managerial recommendations for water utilities. In particular, the findings show that water utilities need to improve service and water quality to increase customers' acceptance of tariff growth. In addition, utilities should invest in customer education and communication activities focusing on specific age groups (e.g., older customers) to enhance their WTP. Finally, communication strategies should reinforce the possible role of liberalization and privatization in supporting infrastructure investments.

Wool Keratin 3D Scaffolds with Light-Triggered Antimicrobial Activity.

Photoactivatable keratin sponges were prepared from protein aqueous solutions by the freeze-drying method, followed by photofunctionalization with two different photosensitizers (PS): Azure A (AzA) and 5,10,15,20-tetrakis [4-(2-N,N,N-trimethylethylthio)-2,3,5,6-tetrafluorophenyl]porphyrin tetraiodide salt (TTFAP). The prepared sponges have a porosity between 49% and 80% and a mean pore size in the 37-80 µm range. As compared to AzA, TTFAP interacts more strongly with the sponges as demonstrated by a lower PS release (6% vs 20%), a decreased swelling ratio (1.6 vs 7.4), and a slower biodegradation rate. Nevertheless, AzA-loaded sponges showed the highest photoactivity, as also demonstrated by their higher antibactericidal activity toward both Gram-positive and Gram-negative bacteria. The obtained results suggest that the antimicrobial photodynamic effect can be finely triggered through a proper selection of the amount and type of photosensitizer, as well as through the irradiation time. Finally, all the prepared sponges support human fibroblast cells growth, while no significant cell viability impairment is observed upon light irradiation.

Development of near-infrared photoactivable phthalocyanine-loaded nanoparticles to kill tumor cells: An improved tool for photodynamic therapy of solid cancers.

Conventional photodynamic therapy has shown to be beneficial in the treatment of a variety of tumors. However, one of its major limitations is the inadequate penetration depth of visible light. In order to overcome this constraint, we developed 80nm poly-methylmethacrylate core-shell fluorescent nanoparticles (FNP) loaded with the photosensitizer tetrasulfonated aluminum phthalocyanine (Ptl). To demonstrate the efficacy of our Ptl@FNP we performed in vitro and in vivo studies using a human prostate tumor model. Our data reveal that Ptl@FNP are internalized by tumor cells, favour Ptl intracellular accumulation, and efficiently trigger cell death through the generation of ROS upon irradiation with 680nm light. When directly injected into tumors intramuscularly induced in SCID mice, Ptl@FNP upon irradiation significantly reduce tumor growth with higher efficiency than the bare Ptl. Collectively, these results demonstrate that the newly developed nanoparticles may be utilized as a delivery system for antitumor phototherapy in solid cancers.

Androgen Receptor Targeted Conjugate for Bimodal Photodynamic Therapy of Prostate Cancer in Vitro.

Prostate cancer (PC) represents the most common type of cancer among males and is the second leading cause of cancer death in men in Western society. Current options for PC therapy remain unsatisfactory, since they often produce uncomfortable long-term side effects, such as impotence (70%) and incontinence (5-20%) even in the first stages of the disease. Light-triggered therapies, such as photodynamic therapy, have the potential to provide important advances in the treatment of localized and partially metastasized prostate cancer. We have designed a novel molecular conjugate (DR2) constituted of a photosensitizer (pheophorbide a, Pba), connected to a nonsteroidal anti-androgen molecule through a small pegylated linker. This study aims at investigating whether DR2 represents a valuable approach for PC treatment based on light-induced production of single oxygen and nitric oxide (NO) in vitro. Besides being able to efficiently bind the androgen receptor (AR), the 2-trifluoromethylnitrobenzene ring on the DR2 backbone is able to release cytotoxic NO under the exclusive control of light, thus augmenting the general photodynamic effect. Although DR2 is similarly internalized in cells expressing different levels of androgen receptor, the AR ligand prevents its efflux through the ABCG2-pump. In vitro phototoxicity experiments demonstrated the ability of DR2 to kill cancer cells more efficiently than Pba, while no dark toxicity was observed. Overall, the presented approach is very promising for further development of AR-photosensitizer conjugates in the multimodal photodynamic treatment of prostate cancer.

HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer still lacking effective treatment options. Chemotherapy in combination with immunotherapy can restrict tumor progression and repolarize the tumor microenvironment towards an anti-tumor milieu, improving clinical outcome in TNBC patients. The chemotherapeutic drug paclitaxel has been shown to induce immunogenic cell death (ICD), whereas inhibitors of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, whose expression is shared in immune regulatory and tumor cells, have been revealed to enhance the anti-tumor immune response. However, poor bioavailability and pharmacokinetics, off-target effects and hurdles in achieving therapeutic drug concentrations at the target tissue often limit the effectiveness of combination therapies. Methods: This work describes the development of novel biomimetic and carrier-free nanobinders (NBs) loaded with both paclitaxel and the IDO1 inhibitor NLG919 in the form of bioresponsive and biomimetic prodrugs. A fine tuning of the preparation conditions allowed to identify NB@5 as the most suitable nanoformulation in terms of reproducibility, stability and in vitro effectiveness. Results and discussion: Our data show that NB@5 effectively binds to HSA in cell-free experiments, demonstrating its protective role in the controlled release of drugs and suggesting the potential to exploit the protein as the endogenous vehicle for targeted delivery to the tumor site. Our study successfully proves that the drugs encapsulated within the NBs are preferentially released under the altered redox conditions commonly found in the tumor microenvironment, thereby inducing cell death, promoting ICD, and inhibiting IDO1.

Corrigendum: HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

[This corrects the article DOI: 10.3389/fchem.2024.1378233.].

Core-shell poly-methylmethacrylate nanoparticles as effective carriers of electrostatically loaded anionic porphyrin.

Among the medical applications of nanoparticles, their usage as photosensitizer (PS) carriers for photodynamic therapy (PDT) has attracted increasing attention. In the present study we explored the morphological and photophysical properties of core-shell PMMA nanoparticles (PMMA-NPs) electrostatically post-loaded with the synthetic, water soluble 5,10,15,20-tetrakis(4-sulphonatophenyl)-porphyrin (TPPS4). pH response and singlet oxygen analyses of differently loaded samples proved the high capability of the PMMA-NPs to shield the PS from the environment, while retaining the PS singlet oxygen production capability. Preliminary in vitro imaging and phototoxicity experiments on HepG2 cells demonstrated the efficacy of the system to trigger photoinduced cell death in the culture.

Sulfonates-PMMA nanoparticles conjugates: a versatile system for multimodal application.

We report herein the viability of a novel nanoparticles (NPs) conjugated system, namely the attachment, based on ionic and hydrophobic interactions, of different sulfonated organic salts to positively charged poly(methylmethacrylate) (PMMA)-based core-shell nanoparticles (EA0) having an high density of ammonium groups on their shells. In this context three different applications of the sulfonates@EA0 systems have been described. In detail, their ability as cytotoxic drugs and pro-drugs carriers was evaluated in vitro on NCI-H460 cell line and in vivo against human ovarian carcinoma IGROV-1 cells. Besides, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) was chosen for NPs loading, and its internalization as bioimaging probe was evaluated on Hep G2 cells. Overall, the available data support the interest for these PMMA NPs@sulfonates systems as a promising formulation for theranostic applications. In vivo biological data strongly support the potential value of these core-shell NPs as delivery system for negatively charged drugs or biologically active molecules. Additionally, we have demonstrated the ability of these PMMA core-shell nanoparticles to act as efficient carriers of fluorophores. In principle, thanks to the high PMMA NPs external charge density, sequential and very easy post-loading of different sulfonates is achievable, thus allowing the preparation of nanocarriers either with bi-modal drug delivery behaviour or as theranostic systems.

Conformational flexibility and absolute stereochemistry of (3R)-3-hydroxy-4-aryl-ß-lactams investigated by chiroptical properties and TD-DFT calculations.

The effect of conformational flexibility on the chiroptical properties of a series of synthetic (3R)-3-hydroxy-4-aryl-ß-lactams of known stereochemistry (1-6) was investigated by means of electronic circular dichroism (ECD) measurements and time-dependent density functional theory (TD-DFT) calculations. The application of the ß-lactam sector rules allowed a correct stereochemical characterization of these compounds, with the exception of a thienyl-substituted derivative (cis-). TD-DFT calculations yielded accurate predictions of experimental ECD spectra and [α](D) values, allowing us to assign the correct absolute configuration to all the investigated compounds. A detailed analysis of the ß-lactam ring equilibrium geometry on optimized conformers identified regular patterns for the arrangement of atoms around the amide chromophore, confirming the validity of the ß-lactam sector rules. However, relevant variations in theoretical chiroptical properties were found for compounds bearing a heterocyclic substituent at C4 or a phenyl substituent at C3, whose conformers deviate from these regular geometric patterns. This behavior explains the failure of the ß-lactam sector rules in cis-. This study showed the importance of conformational flexibility for the determination of chiroptical properties and highlighted the strengths and weaknesses of the different methods for the stereochemical characterization of chiral molecules in solution.

Computer tomography and magnetic resonance for multimodal imaging of fossils and mummies.

The study of fossils and mummies has largely benefited from the use of modern noninvasive and nondestructive imaging technologies and represents a fast developing area. In this review, we describe the emerging role of imaging based on Magnetic Resonance (MR) and Computer Tomography (CT) employed for the study of ancient remains and mummies. For each methodology, the state of the art in paleoradiology applications is described, by emphasizing new technologies developed in the field of both CT, such as micro- and nano-CT, dual-energy and multi-energy CT, and MR, with the description of novel dedicated sequences, radiofrequency coils and gradients. The complementarity of CT and MR in paleoradiology is also discussed, by pointing out what MR provides in addition to CT, with an overview on the state of the art of emerging strategies in the use of CT/MR combination for the study of a sample following a multimodal integrated approach.

Bioactive Keratin and Fibroin Nanoparticles: An Overview of Their Preparation Strategies.

In recent years, several studies have focused their attention on the preparation of biocompatible and biodegradable nanocarriers of potential interest in the biomedical field, ranging from drug delivery systems to imaging and diagnosis. In this regard, natural biomolecules-such as proteins-represent an attractive alternative to synthetic polymers or inorganic materials, thanks to their numerous advantages, such as biocompatibility, biodegradability, and low immunogenicity. Among the most interesting proteins, keratin extracted from wool and feathers, as well as fibroin extracted from Bombyx mori cocoons, possess all of the abovementioned features required for biomedical applications. In the present review, we therefore aim to give an overview of the most important and efficient methodologies for obtaining drug-loaded keratin and fibroin nanoparticles, and of their potential for biomedical applications.

HSA-Binding Prodrugs-Based Nanoparticles Endowed with Chemo and Photo-Toxicity against Breast Cancer.

Exploiting the tumor environment features (EPR effect, elevated glutathione, reactive oxygen species levels) might allow attaining a selective and responsive carrier capable of improving the therapeutic outcome. To this purpose, the in situ covalent binding of drugs and nanoparticles to circulating human serum albumin (HSA) might represent a pioneering approach to achieve an effective strategy. This study describes the synthesis, in vitro and in vivo evaluation of bioresponsive HSA-binding nanoparticles (MAL-PTX2S@Pba), co-delivering two different paclitaxel (PTX) prodrugs and the photosensitizer pheophorbide a (Pba), for the combined photo- and chemo-treatment of breast cancer. Stable and reproducible MAL-PTX2S@Pba nanoparticles with an average diameter of 82 nm and a PTX/Pba molar ratio of 2.5 were obtained by nanoprecipitation. The in vitro 2D combination experiments revealed that MAL-PTX2S@Pba treatment induces a strong inhibition of cell viability of MDA-MB-231, MCF7 and 4T1 cell lines, whereas 3D experiments displayed different trends: while MAL-PTX2S@Pba effectiveness was confirmed against MDA-MB-231 spheroids, the 4T1 model exhibited marked resistance. Lastly, despite using a low PTX-PDT regimen (e.g., 8.16 mg/Kg PTX and 2.34 mg/Kg Pba), our formulation showed to foster primary tumor reduction and curb lung metastases growth in 4T1 tumor-bearing mice, thus setting the basis for further preclinical validations.

Double-Layer Fatty Acid Nanoparticles as a Multiplatform for Diagnostics and Therapy.

Today, public health is one of the most important challenges in society. Cancer is the leading cause of death, so early diagnosis and localized treatments that minimize side effects are a priority. Magnetic nanoparticles have shown great potential as magnetic resonance imaging contrast agents, detection tags for in vitro biosensing, and mediators of heating in magnetic hyperthermia. One of the critical characteristics of nanoparticles to adjust to the biomedical needs of each application is their polymeric coating. Fatty acid coatings are known to contribute to colloidal stability and good surface crystalline quality. While monolayer coatings make the particles hydrophobic, a fatty acid double-layer renders them hydrophilic, and therefore suitable for use in body fluids. In addition, they provide the particles with functional chemical groups that allow their bioconjugation. This work analyzes three types of self-assembled bilayer fatty acid coatings of superparamagnetic iron oxide nanoparticles: oleic, lauric, and myristic acids. We characterize the particles magnetically and structurally and study their potential for resonance imaging, magnetic hyperthermia, and labeling for biosensing in lateral flow immunoassays. We found that the myristic acid sample reported a large r2 relaxivity, superior to existing iron-based commercial agents. For magnetic hyperthermia, a significant specific absorption rate value was obtained for the oleic sample. Finally, the lauric acid sample showed promising results for nanolabeling.

Two Beats One: Osteosarcoma Therapy with Light-Activated and Chemo-Releasing Keratin Nanoformulation in a Preclinical Mouse Model.

Osteosarcoma treatment is moving towards more effective combination therapies. Nevertheless, these approaches present distinctive challenges that can complicate the clinical translation, such as increased toxicity and multi-drug resistance. Drug co-encapsulation within a nanoparticle formulation can overcome these challenges and improve the therapeutic index. We previously synthetized keratin nanoparticles functionalized with Chlorin-e6 (Ce6) and paclitaxel (PTX) to combine photo (PDT) and chemotherapy (PTX) regimens, and the inhibition of osteosarcoma cells growth in vitro was demonstrated. In the current study, we generated an orthotopic osteosarcoma murine model for the preclinical evaluation of our combination therapy. To achieve maximum reproducibility, we systematically established key parameters, such as the number of cells to generate the tumor, the nanoparticles dose, the design of the light-delivery device, the treatment schedule, and the irradiation settings. A 60% engrafting rate was obtained using 10 million OS cells inoculated intratibial, with the tumor model recapitulating the histological hallmarks of the human counterpart. By scheduling the treatment as two cycles of injections, a 32% tumor reduction was obtained with PTX mono-therapy and a 78% reduction with the combined PTX-PDT therapy. Our findings provide the in vivo proof of concept for the subsequent clinical development of a combination therapy to fight osteosarcoma.

Pheophorbide A and Paclitaxel Bioresponsive Nanoparticles as Double-Punch Platform for Cancer Therapy.

Cancer therapy is still a challenging issue. To address this, the combination of anticancer drugs with other therapeutic modalities, such as light-triggered therapies, has emerged as a promising approach, primarily when both active ingredients are provided within a single nanosystem. Herein, we describe the unprecedented preparation of tumor microenvironment (TME) responsive nanoparticles exclusively composed of a paclitaxel (PTX) prodrug and the photosensitizer pheophorbide A (PheoA), e.g., PheoA≅PTX2S. This system aimed to achieve both the TME-triggered and controlled release of PTX and the synergistic/additive effect by PheoA-mediated photodynamic therapy. PheoA≅PTX2S were produced in a simple one-pot process, exhibiting excellent reproducibility, stability, and the ability to load up to 100% PTX and 40% of PheoA. Exposure of PheoA≅PTX2S nanoparticles to TME-mimicked environment provided fast disassembly compared to normal conditions, leading to PTX and PheoA release and consequently elevated cytotoxicity. Our data indicate that PheoA incorporation into nanoparticles prevents its aggregation, thus providing a greater extent of ROS and singlet oxygen production. Importantly, in SK-OV-3 cells, PheoA≅PTX2S allowed a 30-fold PTX dose reduction and a 3-fold dose reduction of PheoA. Our data confirm that prodrug-based nanocarriers represent valuable and sustainable drug delivery systems, possibly reducing toxicity and expediting preclinical and clinical translation.

Keratin nanoparticles and photodynamic therapy enhance the anticancer stem cells activity of salinomycin.

The high rates of aggressiveness, drug resistance and relapse of breast cancer (BC) are mainly attributed to the inability of conventional therapies to equally eradicate bulk differentiated cells and cancer stem cells (CSCs). To improve the effectiveness of BC treatments, we report the in-water synthesis of novel keratin-based nanoformulations, loaded with the CSC-specific drug salinomycin (SAL), the photosensitizer chlorin e6 (Ce6) and vitamin E acetate (SAL/Ce6@kVEs), which combine the capability of releasing SAL with the production of singlet oxygen upon light irradiation. In vitro experiments on BC cell lines and CSC-enriched mammospheres exposed to single or combined therapies showed that SAL/Ce6@kVEs determine synergistic cell killing, limit their self-renewal capacity and decrease the stemness potential by eradication of CSCs. In vivo experiments on zebrafish embryos confirmed the capacity of SAL nanoformulations to interfere with the Wnt/ß-catenin signaling pathway, which is dysregulated in BC, thus identifying a target for further translation into pre-clinical models.

Coating Effect on the 1H-NMR Relaxation Properties of Iron Oxide Magnetic Nanoparticles.

We present a 1H Nuclear Magnetic Resonance (NMR) relaxometry experimental investigation of two series of magnetic nanoparticles, constituted of a maghemite core with a mean diameter dTEM = 17 ± 2.5 nm and 8 ± 0.4 nm, respectively, and coated with four different negative polyelectrolytes. A full structural, morpho-dimensional and magnetic characterization was performed by means of Transmission Electron Microscopy, Atomic Force Microscopy and DC magnetometry. The magnetization curves showed that the investigated nanoparticles displayed a different approach to the saturation depending on the coatings, the less steep ones being those of the two samples coated with P(MAA-stat-MAPEG), suggesting the possibility of slightly different local magnetic disorders induced by the presence of the various polyelectrolytes on the particles' surface. For each series, 1H NMR relaxivities were found to depend very slightly on the surface coating. We observed a higher transverse nuclear relaxivity, r2, at all investigated frequencies (10 kHz ≤ νL ≤ 60 MHz) for the larger diameter series, and a very different frequency behavior for the longitudinal nuclear relaxivity, r1, between the two series. In particular, the first one (dTEM = 17 nm) displayed an anomalous increase of r1 toward the lowest frequencies, possibly due to high magnetic anisotropy together with spin disorder effects. The other series (dTEM = 8 nm) displayed a r1 vs. νL behavior that can be described by the Roch's heuristic model. The fitting procedure provided the distance of the minimum approach and the value of the Néel reversal time (τ ≈ 3.5 ÷ 3.9·10-9 s) at room temperature, confirming the superparamagnetic nature of these compounds.